Compounds for site-enhanced delivery of radionuclides and uses thereof

ABSTRACT

A dihydropyridine⃡pyridinium salt type of redox, or chemical, delivery system for the site-specific and/or site-enhanced delivery of a radionuclide to the brain is provided. A chelating agent capable of chelating with a radionuclide and having a reactive hydroxyl, carboxyl, amino, amide or imide group is coupled to a carrier moiety comprising a dihydropyridine⃡pyridinium salt nucleus and then complexed with a radionuclide to provide a new radiopharmaceutical that, in its lipoidal dihydropyridine form, penetrates the blood-brain barrier (&#34;BBB&#34;) and allows increased levels of radionuclide concentration in the brain, particularly since oxidation of the dihydropyridine carrier moiety in vivo to the ionic pyridinium salt retards elimination from the brain while elimination from the general circulation is accelerated. 
     This radionuclide delivery system is well suited for use in scintigraphy and similar radiographic techniques.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of applicant's copending applicationSer. No. 879,120, filed Mar. 19, 1986, now abandoned, which is acontinuation-in-part of application Ser. No. 632,314, filed July 19,1984, now abandoned. Copending application Ser. No. 879,120 is the U.S.national phase of International PCT Application No. PCT/US85/01334,filed July 15, 1985.

FIELD OF THE INVENTION

The present invention relates to a dihydropyridine⃡pyridinium salt typeof redox, or chemical, delivery system for the site-specific and/orsite-enhanced delivery of a radionuclide to the brain and other organs.More particularly, this invention relates to the discovery that achelating agent capable of chelating with a radionuclide and having areactive hydroxyl, carboxyl, amino, amide or imide group can be coupledto a carrier moiety comprising a dihydropyridine⃡pyridinium salt nucleusand then complexed with a radionuclide to provide a newradiopharmaceutical that, in its lipoidal dihydropyridine form,penetrates the blood-brain barrier ("BBB") and allows increased levelsof radionuclide concentration in the brain, particularly since oxidationof the dihydropyridine carrier moiety in vivo to the ionic pyridiniumsalt retards elimination from the brain while elimination from thegeneral circulation is accelerated.

The present radionuclide delivery system is well suited for use inscintigraphy and similar radiographic techniques.

BACKGROUND OF THE INVENTION

Radiographic techniques such as scintigraphy. and the like, findapplication in biological and medical procedures for diagnosis as wellas research. Scintigraphy involves the use of radiopharmaceuticals;i.e., compounds containing (or labeled with) a radioisotope (i.e.radionuclide) which upon introduction into a mammal become localized inspecific organs, tissue, or skeletal material that are sought to beimaged. When the radiopharmaceutical is so localized, traces, plates, orscintiphotos of the existing distribution of the radionuclide may bemade by various radiation detectors known in the art. The observeddistribution of the localized radionuclide can then be used to detectthe presence of pathological conditions, abnormalities, and the like.Radiopharmaceuticals are thus often referred to as radiodiagnostics.

In many cases, radiopharmaceuticals are prepared using target-specificchelating agents which provide a bridge connecting a radionuclide, suchas a radioactive metal like technetium-99m, or the like, and a materialwhich will temporarily localize in the organ, tissue, or skeletalmaterial which is to be imaged. Typical chelating agents for suchpurposes are: polydentate ligands that form a 1:1 or 2:1ligand:radioactive metal complex; macrocyclic ligands of appropriatering size and preferably where all coordinating atoms are in a planarconfiguration; and bicyclic or polycyclic ligands that can encapsulatethe radioactive metal.

It is a well established fact that the delivery of drugs, includingradiopharmaceuticals, to the brain is often seriously limited bytransport and metabolism factors and, more specifically, by thefunctional barrier of the endothelial brain capillary wall deemed theblood-brain barrier. Site-specific delivery and/or sustained delivery ofdrugs to the brain are even more difficult.

It has been previously suggested to deliver a drug species, specificallyN-methylpyridinium-2-carbaldoxime chloride (2-PAM), into the brain, theactive nucleus of which in and of itself constitutes a quaternarypyridinium salt, by way of the dihydropyridine latentiated prodrug formthereof. Such approach is conspicuously delimited to relatively smallmolecule quaternary pyridinium ring-containing drug species and does notprovide the overall ideal result of brain-specific, sustained release ofthe desired drug, with concomitant rapid elimination from the generalcirculation, enhanced drug efficacy and decreased toxicity. Hence, no"trapping" in the brain of the 2-PAM formed in situ results, andobviously no brain-specific, sustained delivery occurs as anyconsequence thereof: the 2-PAM is eliminated as fast from the brain asit is from the general circulation and other organs. Compare U.S. Pat.Nos. 3,929,813 and 3,962,447; Bodor et al, J. Pharm. Sci., 67, No. 5,pp. 685-687 (1978); Bodor et al, Science, Vol. 190 (1975), pp. 155-156;Shek, Higuchi and Bodor, J. Med. Chem., Vol. 19 (1976), pp. 113-117. Amore recent extension of this approach is described by Brewster,Dissertation Abstracts International, Vol. 43, No. 09, March 1983, p.2910B. It has also been speculated to deliver, e.g., an antitumor agent,into the brain by utilizing a dihydropyridine/pyridinium redox carriermoiety therefor, but this particular hypothesis necessarily entailsderivatizing the dihydropyridine/pyridinium carrier with a substituentitself critically designed to control the release rate of the activedrug species from the quaternary derivative thereof, as well as beingcritically functionally coordinated with the particular chemical andtherapeutic activity/nature of the antitumor drug species itself; Bodoret al, J. Pharm. Sci., supra. See also Bodor, "Novel Approaches for theDesign of Membrane Transport Properties of Drugs", in Design ofBiopharmaceutical Properties Through Prodrugs and Analogs, Roche, E. B.(ed.), APhA Academy of Pharmaceutical Sciences, Washington, D.C., pp.98-135 (1976).

More recently, Bodor et al, Science. Vo. 214, Dec. 18, 1981, pp.1370-1372, have reported on site-specific sustained release of drugs tothe brain. The Science publication outlines a scheme for specific andsustained delivery of drug species to the brain. as depicted in thefollowing Scheme 1: ##STR1## According to the scheme in Science, a drug[D] is coupled to a quaternary carrier [QC]⁺ and the [D-QC]⁺ whichresults is then reduced chemically to the lipoidal dihydro form [D-DHC].After administration of [D-DHC] in vivo, it is rapidly distributedthroughout the body, including the brain. The dihydro form [D-DHC] isthen in situ oxidized (rate constant, k₁) (by the NAD⃡NADH system) to theideally inactive original [D-QC]⁺ quaternary salt which, because of itsionic, hydrophilic character, should be rapidly eliminated from thegeneral circulation of the body, while the blood-brain barrier shouldprevent its elimination from the brain (k₃ >>k₂ ; k₃ >>k₇). Enzymaticcleavage of the [D-QC]⁺ that is "locked" in the brain effects asustained delivery of the drug species [D], followed by its normalelimination (k₅), metabolism. A properly selected carrier [QC]⁺ willalso be rapidly eliminated from the brain (k.sub. 6 >>k₂). Because ofthe facile elimination of [D-QC]⁺ from the general circulation, onlyminor amounts of drug are released in the body (k₃ >>k₄); [D] will bereleased primarily in the brain (k₄ >k₂). The overall result ideallywill be a brain-specific sustained release of the target drug species.

Bodor et al have reported, in Science, their work with phenylethylamineas the drug model, which was coupled to nicotinic acid, then quaternizedto give compounds of the formula ##STR2## which were subsequentlyreduced by sodium dithionite to the corresponding compounds of theformula ##STR3## Testing of the N-methyl derivative in vivo supportedthe criteria set forth in Scheme 1. Bodor et al speculated that varioustypes of drugs might possibly be delivered using the depicted oranalogous carrier systems and indicated that use of N-methylnicotinicacid esters and amides and their pyridine ring-substituted derivativeswas being studied for delivery of amino- or hydroxyl-containing drugs,including small peptides, to the brain. No other possible specificcarriers were disclosed.

Other reports of Bodor et al's work have appeared in The Friday EveningPost, Aug. 14, 1981. Health Center Communications, University ofFlorida, Gainesville, Fla.; Chemical & Engineering News, Dec. 21, 1981,pp. 24-25; and Science News, Jan. 2. 1982, Vol. 121, No. 1, page 7.These publications do not suggest any carrier systems other than thespecific N-methyl and N-benzyl nicotinic acid-type carriers disclosed inthe Science publication. Other classes of drugs as well as a fewspecific drugs are mentioned as possible candidates for derivatization;for example, steroid hormones, cancer drugs and memory enhancers areindicated as targets for possible future work, as are enkephalins, andspecifically, dopamine and testosterone. The publications do not suggesthow to link such drugs to the carrier, except possibly when the drugsare simple structures containing a single NH₂ or, perhaps, simplestructures containing a single OH, of the primary or secondary type, asis the case with phenylethylamine or testosterone. There is, forexample, no suggestion of how one of ordinary skill in the art wouldform a drug-carrier combination when the drug has a more complicatedchemical structure than phenylethylamine, e.g., dopamine or anenkephalin. For further details concerning the work withphenylethylamine, dopamine and testosterone, see also Bodor et al, J.Med. Chem., Vol. 26, March 1983, pp. 313-317; Bodor et al, J. Med.Chem., Vol. 26, April 1983, pp. 528-534; Bodor et al, Pharmacology andTherapeutics, Vol. 19, No. 3, pp. 337-386 (April 1983), and Bodor et al,Science, Vol. 221, July 1983, pp. 65-67.

In view of the foregoing, it is apparent that there has existed anacutely serious, long-standing need for a truly effective, generic butnonetheless flexible, method for the site-specific or sustaineddelivery, or both, of drug species to the brain. This need has beenaddressed in International patent application No. PCT/US83/00725 (filedby UNIVERSITY OF FLORIDA on May 12, 1983 and published underInternational Publication No. WO83/03968 on Nov. 24, 1983), whichprovides such a generic method for site-specific, sustained delivery ofdrugs to the brain utilizing a dihydropyridine⃡pyridinium salt type ofredox carrier system. According to the PCT application, a drug(typically having a reactive --OH, --COOH or --NH₂ group) can be coupledto a dihydropyridine⃡pyridinium carrier; the lipoidal dihydro form of thedrug-carrier system readily crosses the blood-brain barrier; thedihydropyridine moiety is then oxidized in vivo to the ideally inactivequaternary form, which is "locked in" the brain, while it is facilelyeliminated from the general circulation; enzymatic cleavage of the"locked in" quaternary effects a sustained delivery of the drug itselfto the brain, to achieve the desired biological effect. Diagnosticagents such as radiopharmaceuticals are generally disclosed in the PCTapplication as possible candidates for the carrier system, but thesynthetic approach of that application, which utilizes the drug itselfas the starting material, is not desirable when radioactive materials,especially relatively short-lived radionuclides, are involved. Moreover,in the case of radionuclides, the earlier objective of an ideallyinactive form locked in the brain would not achieve the desired result.Thus, a serious need still exists for an effective general method forthe site-specific and/or sustained delivery of a desired radionuclide tothe brain.

SUMMARY OF THE INVENTION

It has now been found that a chemical delivery system based upon adihydropyridine⃡pyridinium salt type redox carrier is uniquely wellsuited for an effective site-specific and/or sustained and/or enhanceddelivery of a radionuclide to the brain or like organ, via novelcarrier-containing radiopharmaceuticals, and novel carrier-containingchelating agents and novel carrier-containing precursors thereto, usefulin the preparation of said radiopharmaceuticals. In one aspect, thepresent invention thus provides novel carrier-containing chelating agentprecusors having the formula

    ○ --[QC.sup.+ ].sub.y mX.sup.-n                     (I)

wherein ○ --is the residue of a chelating agent capable of chelatingwith a metallic radionuclide, said chelating agent having at least onereactive functional group selected from the group consisting of amino,carboxyl, hydroxyl, amide and imide, said functional group being notessential for the complexing properties of said chelating agent, saidresidue being characterized by the absence of a hydrogen atom from atleast one of said reactive functional groups of the chelating agent; yis 1 or 2; [QC⁺ ] is the hydrophilic, ionic pyridinium salt form of adihydropyridine⃡pyridinium salt redox carrier; X⁻ is the anion of apharmaceutically acceptable organic or inorganic acid; n is the valenceof the acid anion; and m is a number which when multiplied by n is equalto y.

In another aspect, the present invention provides novelcarrier-containing chelating agents having the formula

    ○ --[DHC].sub.y                                     (II)

and the non-toxic pharmaceutically acceptable salts thereof, wherein ○-- and y are defined as above, and [DHC] is the reduced, biooxidizable,blood-brain barrier penetrating form of dihydropyridine⃡pyridinium saltredox carrier.

In yet another aspect, the present invention provides, as an effectiveradionuclide delivery system, novel carrier-containingradiopharmaceuticals of the formula

     ○M --[DHC].sub.y                                   (III)

and the non-toxic pharmaceutically acceptable salts thereof, wherein Mis a metallic radionuclide and the remaining structural variables aredefined as before; in other words (III) is the chelated, or complexed,counterpart of (II), formed by complexing the novel carrier-containingchelating agent of formula (II) with a radioactive metal. When aradiopharmaceutical of formula (III) is administered, it readilypenetrates the BBB. Oxidation of (III) in vivo affords the correspondingpyridinium salt of the formula

     ○M --[QC.sup.+ ].sub.y mX.sup.-n                   (IV)

wherein the structural variables are as defined above. Because of itshydrophilic, ionic nature, the formula (IV) substance is "locked-in" thebrain, thus allowing radiographic imaging of the radionuclide present inthe complex (IV). While the quaternary "locked-in" form will graduallycleave to release the carrier moiety and the chelate portion of themolecule, such cleavage will generally occur after the most desirableperiod for radiographic imaging has already passed. It is generallyconsidered most desirable, from the standpoint of patient and techniciansafety, to image the target area as soon as possible afteradministration and to use relatively short-lived radioisotopes. Undersuch circumstances, the "locked-in" quaternary form will likely notdegrade to the non-carrier-containing chelate until after theradioactivity has decayed to a considerable extent. Thus, the presentinvention does not in fact provide a system for delivery and imaging ofpreviously known radiopharmaceuticals: by the time the present deliverysystem degrades to a chelate of a known chelating agent and aradioactive metal, said chelate will generally no longer be sufficientlyradioactive for practical imaging. Moreover, once such degradationoccurs, the known chelate may not be retained in the brain in sufficientamounts to allow imaging thereof. Thus, in contrast to the teachings ofthe Bodor et al publications and the aforementioned PCT application,which emphasize the desirability of an inactive quaternary form lockedin the brain, the present invention provides, and indeed requires, anactive quaternary form locked in the brain in order to allow effectiveradionuclide imaging.

The present chelate/carrier system for radionuclides is characterized byenhanced efficacy and decreased toxicity. Indeed, consistent herewithsystemic toxicity is significantly reduced by accelerating theelimination of the quaternary carrier system from the generalcirculation.

Technetium-99m is a preferred radionuclide for diagnostic purposesbecause of its favorable radiation energy, its relatively shorthalf-life, and the absence of corpuscular radiation, and is preferredfor use in the present invention. Other radionuclides that can be useddiagnostically herein in a chelated form are cobalt-57, gallium-67,gallium-68, indium-111, indium-111m, and the like.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are applicable:

The term "drug" as used herein means any substance intended for use inthe diagnosis, cure, mitigation, treatment or prevention of disease inman or other animal.

The term "lipoidal" as used herein designates a carrier moiety which islipid-soluble or lipophilic.

The expression "non-toxic pharmaceutically acceptable salts" as usedherein generally includes the non-toxic salts of products of theinvention of structures (Il) and (III) hereinabove formed withnon-toxic, pharmaceutically acceptable inorganic or organic acids of thegeneral formula HX. For example, the salts include those derived frominorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic,phosphoric, nitric and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicylic, sulfanilic, fumaric,methanesulfonic, toluenesulfonic and the like. The expression "anion ofa pharmaceutically acceptable organic or inorganic acid" as used herein.e.g. in connection with structures (I) and (IV) above, is intended toinclude anions of such HX acids.

It will be appreciated from the foregoing that a compound of formula(III) may be administered as the free base or in the form of a non-toxicpharmaceutically acceptable salt thereof, i.e. a salt which can berepresented by the formula

     ○M --[DHC].sub.y HX

wherein M, ○ --, [DHC], y and HX are defined as before; and that,regardless of the actual form in which the compound is administered, itwill be converted in vivo to a quaternary salt of formula (IV), theanion X⁻ being present in vivo. It is not necessary that the anion beintroduced as part of the compound administered. Indeed, even when thecompound of formula (III) is used in its salt form, the anion of theformula (IV) compound in vivo is not necessarily the same as thatpresent in the formula (III) compound. In fact, the exact identity ofthe anionic portion of the compound of formula (IV) is immaterial to thein vivo transformation of (III) to (IV).

In the expression "at least one reactive functional group selected formthe group consisting of amine, carboxyl, hydroxyl, amide and imide" asused herein, the designated reactive functional groups have thefollowing meanings:

The word "amino" means any primary or secondary amino function, i.e.--NH₂ or --NHR where R is typically C₁ -C₇ alkyl or is a portion of thechelating agent residue itself. The secondary amino function is alsorepresented herein as --NH--, particularly since the exact identity ofthe R portion of --NHR is immaterial, just so long as it does notprevent the formation of the chelating agent residue and its linkage tothe carrier moiety or otherwise interfere with the objects of thisinvention.

The word "carboxyl" means a --COOH function.

The word "hydroxyl" means an --OH function.

The word "amide" means a carbamoyl (--CONH₂) or substituted carbamoyl(--CONHR, where R is typically C₁ -C₇ alkyl) functional group. The--CONHR group may also be represented herein as --CONH--, since theexact identity of the R portion of --CONHR is immaterial, just so longas it does not prevent the formation of the chelating agent residue andits linkage to the carrier moiety or otherwise interfere with theobjects of this invention.

The word "imide" means a functional group having the structure ##STR4##that is, the structure which characterizes imides (i.e. compounds suchas succinimide, pathalimide and so forth).

The expression "said functional group being not essential for thecomplexing properties of said chelating agent" is believed to beself-explanatory. Any functional group in the chelating agent which canbe linked to the carrier moiety without destroying the chelating agent'sability to complex with the radionuclide is considered herein to be notessential for complexing properties. On the other hand, derivation of afunctional group which would lead to a carrier-containing structurewhich would be incapable of complexing with a radionuclide is not withinthe ambit of this invention.

In accord with the present invention, the sustained delivery of aradionuclide to the brain in sufficient concentrations for radioimagingcan be effected with much lower concentrations in the peripheralcirculation and other tissues. The present invention of course willallow such imaging of any other organs or glands in which sufficientradioactivity accumulates. Thus, for example, it is expected that thequaternary form (IV) which is locked in the brain will be locked in thetestes as well. See the aforementioned PCT application.

The novel radionuclide delivery system of this invention begins with thepreparation of the novel carrier-containing chelating agent precursorsof formula (I). The preparation of those precursors will be tailored tothe particular chelating portion and carrier portion to be combined, andespecially to the nature of the chemical bond between them, e.g. whetherthe linkage is an ester or amide linkage, as well as to the presence orabsence of other reactive functional groups (amino, mercapto, carboxyl,hydroxy) in either the chelating or carrier portion. Typically, if suchother reactive groups are present, they are found in the chelatingportion. In any event, when such groups are present and it is desired toprotect them, a step that introduces appropriate protecting groups canbe incorporated at a suitable stage of the synthetic pathway. Protectivegroups are well known in the art and include t-butoxycarbonyl for aminogroups, N-methyleneacetamido for mercaptans, and N-hydroxysuccinimidylfor carboxyl groups. Acyl or carbonate groups are typically utilized toprotect alcohol hydroxyls. When carbonate protecting groups are desired,the step of introducing the protecting groups will involve reacting thealcohol with a halocarbonate of the type ROCOCl or ROCOBr (formed byreaction of ROH with COCl₂ or COBr₂, R typically being loweralkyl). Foracyl protecting groups, the alcoholic hydroxyl is reacted with an acylhalide RCl or RBr, R being --COCH₃ or --COC(CH₃)₃. Yet other reactionschemes and reactants will be readily apparent to those skilled in theart as will the appropriate means for removing such protective groupsafter they have achieved their function and are no longer needed.

In forming the precursors of formula (I), at least one --COOH, --OH,primary or secondary amino, amide or imide group in a chelating agentwill be bonded to [QC⁺ ], the hydrophilic, ionic pyridinium salt form ofa dihydropyridine⃡pyridinium salt redox carrier.

It will be appreciated that by [QC⁺ ] there is intended any non-toxiccarrier moiety comprising, containing or including the pyridiniumnucleus, whether or not a part of any larger basic nucleus, and whethersubstituted or unsubstituted, the only criterion therefor being capacityfor chemical reduction to the corresponding dihydropyridine form [DHC],BBB-penetration of [DHC] and in vivo oxidation of [DHC] back to thequaternary pyridinium salt carrier moiety [QC⁺ ].

As aforesaid, the ionic pyridinium salt radiopharmaceutical/carrierentity of formula (IV) which results from in vivo oxidation of thedihydropyridine form (III) is prevented from efflux from the brain,while elimination from the general circulation is accelerated.Radioimaging of the radionuclide present in the "locked in" formula (IV)quaternary allows observation of the distribution of the localizedradionuclide for diagnosis of pathological conditions, abnormalities,etc. Subsequently, the coupling between the particular radioactivespecies ○M --and the quaternary carrier [QC]⁺ is likely metabolicallycleaved which results in facile elimination of the carrier moiety [QC⁺].

Coupling between the chelate moiety and the quaternary carrier can be asimple direct chemical bond, e.g., an amide bond or ester bond, or anyother like bond, or same can even be comprised of a linking group orfunction as is illustrated in the Examples or the ethylenediamine groupillustrated in Schemes 3 and 4. Nonetheless, the bond is intended to be,and is hereby defined as, inclusive of all such alternatives.

Eventual cleavage of the formula (IV) quaternary with facile eliminationof the carrier moiety [QC⁺ ] is characteristically an enzymatic orchemical cleavage,. e.g., by an amidase, albeit any type in braincleavage which might result, whether enzymatic, metabolic or otherwise,of course remains within the ambit of this invention.

The many different dihydropyridine⃡pyridinium salt redox carrier moietiesillustrated for use hereinbelow are merely exemplary of the many classesof carriers contemplated by this invention. While the following list ofcarrier classes is not meant to be exhaustive (and, indeed yet othercarrier classes are illustrated hereinbelow as well as in theaforementioned PCT application, PCT/US83/00725), the following majorclasses of quaternaries and the corresponding dihydro forms are primeexamples of the moieties encompassed hereby:

(1) For linkage to a chelating agent having at least one --NH₂, --NH--or --OH functional grouping, replacing a hydrogen atom from at least oneof said functional groupings with one of the following [QC⁺ ] groupings:##STR5## wherein the alkylene group can be straight or branched and cancontain 1 to 3 carbon atoms; R_(o) is a radical identical to thecorresponding portion of a natural amino acid; p is 0, 1 or 2, providedthat, when p is 2, then the alkylene groups can be the same or differentand the R_(o) radicals can be the same or different: R₁ is C₁ -C₇ alkyl,C₁ -C₇ haloalkyl or C₇ -C₁₀ aralkyl; R₃ is C₁ to C₃ alkylene; X is--CONR'R" wherein R' and R", which can be the same or different, areeach H or C₁ -C₇ alkyl, or X is --CH═NOR'" wherein R'" is H or C₁ -C₇alkyl; the carbonyl-containing groupings in formulas (a) and (c) and theX substituent in formula (b) can each be attached at the 2, 3 or 4position of the pyridinium ring; the carbonyl-containing groupings informulas (d) and (f) and the X substituent in formula (e) can each beattached at the 2, 3 or 4 position of the quinolinium ring; and thecarbonyl-containing groupings in formulas (g) and (j) and the Xsubstituent in formula (h) can each be attached at the 1, 3 or 4position of the isoquinolinium ring;

(2) For the linkage to a chelating agent having at least one --COOHfunctional grouping, replacing a hydrogen atom from at least one of said--COOH groupings with one of the following [QC⁺ ] groupings:

(a) When there are one or two --COOH groups to be derivatized: ##STR6##wherein the alkylene group can be straight or branched and can contain 1to 3 carbon atoms; R_(o) is a radical identical to the correspondingportion of a natural amino acid; p is 0, 1 or 2, provided that, when pis 2, then the alkylene groups can be the same or different and theR_(o) radicals can be the same or different; Z'is C₁ -C₈ straight orbranched alkylene, preferably C₁ -C₃ straight or branched alkylene; Q is--O-- or --NH--; R₁ is C₁ -C₇ alkyl, C₁ -C₇ haloalkyl or C₇ -C₁₀aralkyl; R₃ is C₁ -C₃ alkylene; X is --CONR'R" wherein R' and R", whichcan be the same or different, are each H or C₁ -C₇ alkyl, or X is--CH═NOR'" wherein R'" is H or C₁ -C₇ alkyl; the X substituent informula (ii) and the carbonyl-containing groupings in formulas (i) and(iii) can each be attached at the 2, 3 or 4 position of the pyridiniumring; the X substituent in formula (v) and the carbonyl-containinggroupings in formulas (iv) and (vi) can each be attached at the 2, 3 or4 position of the quinolinium ring; and the X substituent in formula(viii) and carbonyl-containing groupings in formulas (vii) and (ix) caneach be attached at the 1, 3 or 4 position of the isoquinolinium ring;

(b) Alternatively, when there is only one --COOH group to bederivatized: ##STR7## wherein .BHorizBrace. is the skeleton of a sugarmolecule; n^(iv) is a positive integer equal to the total number of --OHfunctions in the sugar molecule from which said skeleton is derived;n^(v) is a positive integer one less than the total number of --OHfunctions in the sugar molecule from which said skeleton is derived;each A in each of structures (xii), (xiii) and (xiv) can independentlybe hydroxy or D', D' being the residue of a chelating agent containingone reactive --COOH functional group, said residue being characterizedby the absence of a hydrogen atom from said --COOH functional group insaid chelating agent; and each R'₄ in each of structures (x) and (xi)can independently be hydroxy, ##STR8## wherein the alkylene group can bestraight or branched and can contain 1 to 3 carbon atoms; R_(o) is aradical identical to the corresponding portion of a natural amino acid;P is 0, 1 or 2, provided that, when P is 2, then the alkylene groups canbe the same or different and the R_(o) radicals can be the same ordifferent; D' is defined as with structure (xii), (xiii) and (xiv); R₁is C₁ -C₇ alkyl, C₁ -C₇ haloalkyl or C₇ -C₁₀ aralkyl; and the depictedcarbonyl-containing groupings can be attached at the 2, 3 or 4 positionof the pyridinium or quinolinium ring, or at the 1, 3 or 4 position ofthe isoquinolinium ring; with the proviso that at least one R'₄ in eachof structures (x) and (xi) is ##STR9## wherein alkylene, R_(o), p and R₁and the position of the carbonyl-containing groupings are defined asabove; and with the further proviso that when more than one of the R'₄radicals in a given compound are the aforesaid carbonyl-containinggroupings, then all such carbonyl-containing groupings in said compoundare identical;

(3) For linkage to a chelating agent having at least one --NH--functional group which is part of an amide or imide structure or atleast one low pKa primary or secondary amine functional group, replacinga hydrogen atom from at least one of said functional groupings with oneof the following [QC⁺ ] groupings: ##STR10## wherein the alkylene groupcan be straight or branched and can contain 1 to 3 carbon atoms; R_(o)is a radical identical to the corresponding portion of a natural aminoacid; p is 0, 1 or 2, provided that, when p is 2, then the alkylenegroups can be the same or different and the R_(o) radicals can be thesame or different; R₁ is C₁ -C₇ alkyl, C₁ -C₇ haloalkyl or C₇ -C₁₀aralkyl; R is hydrogen, C₁ -C₇ alkyl, C₃ -C₈ cycloalkyl, C₁ -C₇haloalkyl, furyl, phenyl, or phenyl substituted by one or more halo,lower alkyl, lower alkoxy, carbamoyl, lower alkoxycarbonyl, loweralkanoyloxy, lower haloalkyl, mono(lower alkyl)carbamoyl, di(loweralkyl)carbamoyl, lower alkylthio, lower alkylsulfinyl or loweralkylsulfonyl; R₃ is C₁ to C₃ alkylene; X is --CONR'R" wherein R' andR", which can be the same or different, are each H or C₁ -C₇ alkyl, or Xis --CH═NOR'" wherein R'" is H or C₁ -C₇ alkyl; the carbonyl-containinggroupings in formulas (k) and (m) and the X substituent in formula (1)can each be attached at the 2, 3 or 4 position of the pyridinium ring;the carbonyl containing groupings in formulas (n) and (p) and the Xsubstituent in formula (o) can each be attached at the 2, 3 or 4position of the quinolinium ring; and the carbonyl-containing groupingsin formulas (q and (s) and the X substituent in formula (r) can each beattached at the 1, 3 or 4 position of the isoquinolinium ring.

Here and throughout this application, the expression "C₁ -C₇ haloalkyl"means C₁ -C₇ alkyl substituted by one or more halogen atoms. Also hereand throughout this application, the alkyl radicals, including alkyl andalkylene portions of other radicals, can be straight or branched unlessotherwise specified.

The expression "R_(o) is a radical identical to the correspondingportion of a natural amino acid" is believed to be self-explanatory.Thus, for example. R_(o) can be hydrogen, as in glycine; methyl, as inalanine; --CH(CH₃)₂, as in valine; --CH₂ -CH(CH₃)₂, as in leucine;##STR11## --CH₂ OH, as in serine; --CHOH--CH₃, as in threonine; --(CH₂)₂--SCH₃, as in methionine; --CH₂ --CONH₂, as in asparagine; --CH₂ CH₂--CONH₂, as in glutamine; ##STR12## --CH₂ SH, as in cysteine; --CH₂COOH, as in aspartic acid; and --CH₂ CH₂ COOH, as in glutamic acid. Theexpression "natural amino acid" as used herein does not encompass dopaor L-DOPA. Preferred amino acids encompassed by the R_(o) term includeglycine, alanine, valine, leucine, phenylalanine, isoleucine,methionine, asparagine and glutamine.

The dihydro forms [DHC] corresponding to the aforementioned quaternariesare as follows:

(1') For Group (1) above: ##STR13## wherein the alkylene group can bestraight or branched and can contain 1 to 3 carbon atoms; R_(o) is aradical identical to the corresponding portion of a natural amino acid;p is 0, 1 or 2, provided that, when p is 2, then the alkylene groups canbe the same or different and the R_(o) radicals can be the same ordifferent; the dotted line in formulas (a'), (b') and (c') indicates thepresence of a double bond in either the 4 or 5 position of thedihydropyridine rin9; the dotted line in formulas (d'), (e') and (f')indicates the presence of a double bond in either the 2 or 3 position ofthe dihydroquinoline ring; R₁ is C₁ -C₇ alkyl, C₁ -C₇ haloalkyl or C₇-C₁₀ aralkyl; R₃ is C₁ to C₃ alkylene; X is --CONR'R", wherein R' andR", which can be the same or different, are each H or C₁ -C₇ alkyl, or Xis --CH═NOR'" wherein R'" is H or C₁ -C₇ alkyl; the carbonyl-containinggroupings in formulas (a') and (c') and the X substituent in formula(b') can each be attached at the 2, 3 or 4 position of thedihydropyridine ring the carbonyl-containing groupings in formulas (d')and (f') and the X substituent in formula (e') can each be attached at2, 3 or 4 position of the dihydroquinoline ring; and thecarbonyl-containing groupings in formulas (g') and (j') and the Xsubstituent in formula (h') can each be attached at the 1, 3 or 4position of the dihydroisoquinoline ring;

(2') For Group (2) (a) above: ##STR14## wherein the alkylene group canbe straight or branched and can contain 1 to 3 carbon atoms; R_(o) is aradical identical to the corresponding portion of a natural amino acid;p is 0, 1 or 2, provided that, when p is 2, then the alkylene groups canbe the same or different and the R_(o) radicals can be the same, ordifferent; the dotted line in formulas (i'), (i') and (iii') indicatesthe presence of a double bond in either the 4 or 5 position of thedihydropyridine ring; the dotted line in formulas (iv'), (v') and (vi')indicates the presence of a double bond in either the 2 or 3 position ofthe dihydroquinoline ring; Z' is C₁ -C₈ straight or branched alkylene,preferably C₁ -C₃ straight or branded alkylene; Q is --O-- or --NH--; R₁is C₁ -C₇ alkyl, C₁ -C₇ haloalkyl or C₇ -C₁₀ aralkyl; R₃ is C₁ -C₃alkylene; X is --CONR'R" wherein R' and R", which can be the same ordifferent, are each H or C₁ -C₇ alkyl, or X is --CH═NOR'" wherein R'" isH or C₁ -C₇ alkyl; the X substituent in formula (ii') and thecarbonyl-containing grouping in formulas (i') and (iii') can each beattached at the 2,3 or 4 position of the dihydropyridine ring; the Xsubstituent in formula (v') and the carbonyl-containing grouping informulas (iv') and (vi') can each be attached at the 2, 3 or 4 positionof the dihydroquinoline ring; and the X substituent in formula (viii')and the carbonyl-containing groupings in formulas (vii') and (ix') caneach be attached at the 1, 3 or 4 position of the dihydroisoquinolinering;

(3') For Group (2) (b) above: ##STR15## wherein the alkylene group canbe straight or branched and can contain 1 to 3 carbon atoms; R_(o) is aradical identical to the corresponding portion of a natural amino acid;p is 0, 1 or 2, provided that, when p is 2, then the alkylene groups canbe the same or different and the R_(o) radicals can be the same ordifferent; the dotted line in formula (xii') indicates the presence of adouble bond in either the 4 or 5 position of the dihydropyridine ring;the dotted line in formula (xiii') indicates the presence of a doublebond in either the 2 or 3 position of the dihydroquinoline ring; is theskeleton of a sugar molecule; n^(iv) is a positive integer equal to thetotal number of --OH functions in the sugar molecule from which saidskeleton is derived; n^(v) is a positive integer one less than the totalnumber of --OH functions in the sugar molecule from which said skeletonis derived; each A in each of structures (xii'), (xiii'), (xiv') and(xiv") can independently be hydroxy or D', D' being the residue of achelating agent containing one reactive --COOH functional group, saidresidue being characterized by the absence of a hydrogen atom from said--COOH functional group in said chelating agent; and each R₄ in each ofstructures (x') and (xi') can independently be hydroxy, ##STR16##wherein the alkylene group can be straight or branched and can contain 1to 3 carbon atoms; R_(o) is a radical identical to the correspondingportion of a natural amino acid; p is 0, 1 or 2, provided that, when pis 2, then the alkylene groups can be the same or different and theR_(o) radicals can be the same or different; the dotted line is definedas with structures (xii') and (xiii'); D' is defined as with structures(xii'), (xiii'), (xiv') and (xiv"); R₁ is C₁ -C₇ alkyl, C₁ -C₇ haloalkylor C₇ -C₁₀ aralkyl; and the depicted carbonyl groupings can be attachedat the 2, 3 or 4 position of the pyridinium or quinolinium ring or,except where otherwise specified, at the 1, 3 or 4 position of theisoquinolinium ring; with the proviso that at least one R₄ in each ofstructures (x') and (xi') is ##STR17## wherein alkylene, R_(o), p, R₁,the dotted lines and the position of the carbonyl-containing groupingsare defined as above; and with the further proviso that when more thanone of the R₄ radicals in a given compound are the aforesaidcarbonyl-containing groupings, then all such carbonyl-containinggroupings in said compound are identical;

(4') For Group (3) above: ##STR18## wherein the alkylene group can bestraight or branched and can contain 1 to 3 carbon atoms; R_(o) is aradical identical to the corresponding portion of a natural amino acid;p is 0, 1 or 2, provided that, when p is 2, then the alkylene groups canbe the same or different and the R_(o) radicals can be the same ordifferent; R is hydrogen, C₁ -C₇ alkyl, C₃ -C₈ cycloalkyl, C₁ -C₇haloalkyl, furyl, phenyl, or phenyl substituted by one or more halo,lower alkyl, lower alkoxy, carbamoyl, lower alkoxycarbonyl, loweralkanoyloxy, lower haloalkyl, mono(lower alkyl)carbamoyl, di(loweralkyl)carbamoyl, lower alkylthio, lower alkylsulfinyl or loweralkylsulfonyl; the dotted line in formulas (k'), (l') and (m') indicatesthe presence of a double bond in either the 4 or 5 position of thedihydropyridine ring; the dotted line in formulas (n'), (o') and (p')indicates the presence of a double bond in either the 2 or 3 position ofthe dihydroquinoline ring; R₁ is C₁ -C₇ alkyl, C₁ -C₇ naloalkyl or C₇-C₁₀ aralkyl; R₃ is C₁ to C₃ alkylene; X is --CONR'R", wherein R' andR", which can be the same or different, are each H or C₁ -C₇ alkyl, or Xis --CH--NOR'", wherein R'" is H or C₁ -C₇ alkyl; thecarbonyl-containing groupings in formulas (k') and (m') and the Xsubstituent in formula (l') can each be attached at the 2, 3 or 4position of the dihydropyridine ring; the carbonyl-containing groupingsin formulas (n') and (p') and the X substituent in formula (o') can eachbe attached at the 2, 3 or 4 position of the dihydroquinoline ring; andthe carbonyl-containing groupings in formulas (q') and (s') and the Xsubstituent in formula (r') can each be attached at the 1, 3 or 4position of the dihydroisoquinoline ring.

The presently preferred dihydropyridine⃡pyridinium salt redox carriermoieties of this invention are those wherein p is 0 or 1, mostpreferably 0; alkylene, when present (i.e. p=1 or 2). is --CH₂ --;R_(o), when present(i.e. p=1 or 2), is H, --CH₃, --CH(CH₃)₂, --CH₂--CH(CH₃)₂, ##STR19## --CH₂)₂ --SCH₃, --CH₂ --CONH₂ or --CH₂ CH₂ --CONH₂; R₁, when present, is --CH₃ ; R₃, when present, is --CH₂ CH₂ --; X,when present, is --CONH₂ ; the depicted carbonyl-containing groupings informulas (a) and (c) and the X substituent in formula (b) are attachedat the 3-position; the depicted carbonyl-containing groupings informulas (d) and (f) and the X substituent in formula (e) are attachedat the 3-position; the depicted carbonyl-containing groupings informulas (g) and (j) and the X substituent in formula (h) are attachedat the 4-position; Z', when present, is C₂ or C₃ straight or branchedalkylene; Q, when present, is --NH--; the X substituent in formulas (ii)and (v) and the depicted carbonyl-containing groupings in formulas (i),(iii), (iv) and (vi) are attached at the 3-position; the X substituentin formula (viii) and the depicted carbonyl-containing groupings informulas (vii) and (ix) are attached at the 4 -position; and thedepicted carbonyl-containing groupings encompassed by formulas (x),(xi), (xii), (xiii) and (xiv) are in the 3-position of the pyridinium orquinolinium ring and in the 4-position of the isoquinolinium ring; allR'₄ 's in structures (x) and (xi) are --OH except for the one R₄ in eachstructure which must be the carrier moiety; all A's in structures (xii),(xiii) and (xiv) are --OH; is the skeleton of a glucose molecule; R informulas (k), (l) and (m) is hydrogen, methyl or CCl₃ ; and the depictedcarbonyl-containing groupings in formulas (k) through (s)are in the3-position of the pyridinium or quinolinium ring and in the 4-positionof the isoquinolinium ring; and the corresponding dihydro moieties.

Especially preferred dihydropyridine⃡pyridinium salt redox carriermoieties are quaternaries of Group (1), structures (a), (b), (d), (e),(g) and (h); those of Group (2), structures (i), (ii), (iv), (v), (vii),(viii), (x) and (xii); and those of Group 3, structures (k), (l), (n),(o), (q) and (r); and the corresponding dihydro forms, most especiallywhen they contain the preferred structural variables identified in thepreceding paragraph.

The following synthetic schemes illustrate various approaches to thepreparation of the carrier-containing chelating agent precursors offormula (I), to the corresponding carrier-containing chelating agents offormula (II) and to the corresponding carrier-containingradiopharmaceuticals of formula (III). Also shown are the corresponding"locked in" quaternaries of formula (IV) formed by in vivo oxidation ofthe formula (III) chelates, said formula (IV) quaternaries being theprimary localized materials whose radionuclide content is imaged byradiation detection means. ##STR20##

Thus, Scheme 1 above illustrates a typical synthetic route for compoundsin which the linkage between the carrier and chelate portions is througha --COOH function in the chelating agent. In the first step, the alcoholreactant can be represented generally as HO--Z'--I wherein Z' is C₁ -C₈straight or branched alkylene; in the second step, the depictedreactant, nicotinamide, could be readily replaced with picolinamide,isonicotinamide, 3-quinolinecarboxamide, 4-isoquinolinecarboxamide orthe like. (3-Quinolinecarboxamide and 4-isoquinolinecarboxamide can beprepared in known manner, e.g. by treating the corresponding acids withammonia.) Other process variations will be apparent to those skilled inthe art, particularly from the teachings of the aforementionedInternational Application PCT/US83/00725.

One such alternate approach to Scheme 1 is depicted in Scheme 2. In thefirst step of Scheme 2, the alcohol reactant (prepared by reacting2-iodoethanol with nicotinamide) could contain a shorter or longeralkylene bridge (C₁ -C₈) than shown and the pyridinium portion could bereplaced with an equivalent pyridinium carrier, prepared in analogousfashion. Thus, for example, in the first step, an alcohol of the formula##STR21## wherein n=1-8, preferably 1-3, can be reacted with 7 or other--COOH-containing chelating agent or precursor thereof. Alternatively,an alcohol of the formula ##STR22## (prepared by reacting nicotinic acidwith 1,2-propylene glycol in the presence of dicyclohexylcarbodiimide)or a position isomer or homologue thereof or corresponding derivative ofa quinolinecarboxylic acid or an isoquinolinecarboxylic acid can bequaternized, e.g. by reaction with methyl iodide, and used in place ofthe alcohol reactant shown in Scheme 2. As yet another variation,bromoglucose can be reacted with nicotinamide, picolinamide orisonicotinamide or appropriate quinolinecarboxamide orisoquinolinecarboxamide to afford a starting alcohol of the formula##STR23## which can be used in place of the alcohol reactant used in thefirst step of Scheme 2. Still other variations would include reactingnicotinic acid or other suitable pyridine-ring containing acid with anappropriate di- or polyhydroxy compound such as ethylene glycol,propylene glycol, inositol or a simple sugar, linking the resultantintermediate via its free hydroxy group(s) to the carboxylic acidfunction of the chelating agent or the precursor thereof, and thenquaternizing that intermediate.

Schemes 3 and 4 above are illustrative of the type of procedure utilizedto prepare compounds in which the linkage between the carrier andchelate portions is through an --NH₂ or --OH function in the chelatingagent or precursor thereof. The activated ester of nicotinic acid, 16,can of course be replaced with another activated ester of that or asimilar pyridine-ring containing acid. Equivalent activated esters, e.g.an ester in which the ##STR24## is replaced with (especiallyp-nitrophenyl) will be apparent to those skilled in the art. Thepreparation of such esters proceeds according to known procedures, e.g.by reacting the acid chloride or anhydride or the acid in the presenceof DCC with N- hydroxysuccinimide or other alcohol, then quaternizingthe product, e.g. with methyl iodide or dimethylsulfate.

Scheme 5 illustrates another possible approach when the linkage betweenthe carrier and chelate portions is through an --OH function in thechelating agent or its precursor. The first step in this sequence isdescribed in Fritzberg U.S. Pat. No. 4,444,690; the resultant ethylester 30 is then reduced to the corresponding alcohol, using anappropriate reducing agent, e.g. LiAlH₄. The reduction thus introduces a--CH₂ OH function in place of the acid function in 7. Other --COOHcontaining chelating agents or their precursors can be similarlyconverted to the corresponding --CH₂ OH containing compounds, which canthen be derivatized to the carrier-containing moieties as generallydescribed hereinabove. One such derivatization is shown in Scheme 5. Theconversions 31→32→33→34 parallel reactions shown in Scheme 2 hereinabovewell as in the Fritzberg Patent. The carrier-containing moiety canreadily be introduced into the structure after obtaining 34 by a varietyof methods, e.g. by use of the activated quaternized ester 17 used inSchemes 3 and 4 or other activated ester;or by reaction with bromoacetylchloride, followed by reaction with nicotinamide, isonicotinamide,3-quinolinecarboxamide, picolinamide, 4-isoquinolinecarboxamide or thelike to form 36 or similar derivative. Subsequent reduction to thedihydropyridine form as described herein and in InternationalApplication No. PCT/US83/00725 can be performed separately, or, moreconveniently, can be accomplished at the same time as reduction oftechnetium to an appropriate oxidation state.

Scheme 6 illustrates a method of particular use when the linkage betweenthe carrier and chelate portions is through an --NH--function which ispart of an amide or imide or a very low pKa primary or secondary amine.Conversion of an ester group to the corresponding amide is accomplishedwith excess ammonium. Then the chelating agent precursor 42 having a--CONH₂ funtional group is subjected to N-hydroxyalkylation, e.g. byreaction with an aldehyde [e.g. formaldehyde, benzaldehyde, acetaldehydeor choral(Cl₃ CCHO)]thus, for example, in the case of chloral, the CONH₂group becomes a ##STR25## function and thus forms a suitable bridginggroup. The resultant compound is then subjected to any method describedherein or in the aforementioned PCT application for linking the carrierto an --OH function. One such method, i.e. reacting the alcohol withnicotinic acid in the presence of dicyclohexylcarbodiimide, is shown inScheme 6.

Scheme 7 is illustrative of a process in which the --NH--group to whichthe carrier is to be linked is part of an imide structure. The earlieststeps of this Scheme are described in the aforementioned Fritzbergpatent. Then, 51 is reacted with excess ammonia to form thecorresponding succinimide which, when heated, loses ammonia to give thesuccinimide 52. That intermediate is then reacted with an aldehyde. asgenerally described in the preceding paragraph. and the resulting --OHcontaining group then derivatized, also as described previously.

Scheme 8 illustrates yet another alternate to Schemes 1 and 2;3,4-diaminobenzoic acid is disclosed as a starting material forchelating agents in the Fritzberg patent. Scheme 8 follows the reactionsequence of Scheme 2 and could be varied in any of the many waysdescribed in conjunction with Scheme 2 hereinabove. Moreover, 59 couldalternatively be subjected to the reactions shown in Schemes 5 and 6and/or discussed in connection with those Schemes; i.e. the --COOH groupcould be converted to a --CH₂ OH or a --CONH₂ group and then derivatizedas shown in and discussed with respect to those Schemes.

Schemes 9, 12 and 14 above illustrate typical conversion of a carboxylicacid ester group to the corresponding amide (--CONH₂); reduction of theamide function to the corresponding amine (--CH₂ NH₂); reaction of the--NH₂ group with an activated ester of nicotinic acid; quaternizationwith methyl iodide; and reduction of the resultant quaternary of formula(I) to the corresponding dihydro of formula (11), or conversion of (I)directly to the formula (III) radiopharmaceutical. These processes canbe varied as discussed in conjunction with Schemes 3, 4 and 5 above.

Schemes 10 and 15 above illustrate typical conversion of an alcohol(--CH₂ OH), which may be obtained from the corresponding carboxylic acidester, to the corresponding nicotinoyl ester; reaction of the esterderivative with methyl iodide to afford the desired formula (I)quaternary; and reduction to the corresponding formula (II) dihydro orconversion directly to the corresponding formula (III)radiopharmaceutical. For Process variations, see the discussion ofSchemes 3, 4 and 5 hereinabove.

In Scheme 11 above, there is shown a typical method for introducing alonger alkylene chain between an atom which is involved in forming thechelate structure and a pendant NH₂ group which is to be coupled to thecarrier moiety. As depicted in this scheme, a secondary amino group >NHis reacted with a haloalkamide, e.g. BrCH₂ CONH₂, replacing the hydrogenof the >NH with --CH₂ CONH₂. Reduction of the amide affords thecorresponding >NCH₂ CH₂ NH₂ compound. That amine can then be reactedwith an activated ester of nicotinic acid, followed by quaternizationand reduction is in the other schemes. For variations, see in particularSchemes 3, 4 and 5 above.

Schemes 13 and 16 illustrate yet other methods for lengthening thealkylene chain, the chain here being interrupted by one or more oxygenatoms. Thus, a --CH₂ OH group is typically convertcd to thecorresponding lithium salt and then reacted with an iodoalkanol, e.g.ICH₂ CH₂ OH, to convert the --CH₂ O--Li⁺ group to a --CH₂ OCH₂ CH₂ OHgroup. [Obviously, the chain could be lengthened by utilizing alonger-chain iodoalkanol, or by repeating the two steps just described(in which case additional intervening oxygen atoms would beintroduced.)] The --CH₂ OCH₂ CH₂ OH group is then converted to thecorresponding nicotinic acid ester, which is then quaternized to formthe desired quaternary salt. Again, the reaction schemes can be variedas discussed with reference to Schemes 3, 4 and 5 hereinabove.

In Scheme 17 above, reaction of an --NH₂ group with an activated esterof nicotinic acid, followed by quaternization, is shown. The resultantformula (I) quaternary is then reduced as shown in the other schemes.

Many of the earliest steps in the reaction schemes depicted aboveparallel reactions described in Fritzberg U.S. Patent No. 4,444,690.See, for example, the conversion of 7 to 30 in Scheme 10; the conversionof 30 to 40 to 41 in Scheme 12; the conversion of 111 to 112 to 113 to114 in Scheme 13; and so on.

Scheme 18 above, like Scheme 11 which has already been discussed, showsanother typical method for introducing a longer alkylene chain betweenthe nitrogen atoms. Here the secondary amino group >NH is converted tothe corresponding >NCH₂ CH₂ CH₂ NH₂ group. The resultant amine can thenbe reacted with an activated ester of nicotinic acid, followed byquaternization and reduction as in the other schemes. As a preferredalternative in this and many of the other reaction schemes depictedherein the quaternary chelating agent Precursor of the invention can beprepared directly from reaction of the corresponding amine with aquaternized activated ester of nicotinic acid. Other variations will beapparent, e.g. from Schemes 3, 4 and 5 above.

Scheme 19 represents an alternate approach to the derivatives resultingfrom Scheme 1. Obviously, this scheme could be varied in a number ofways, most notably in the fourth step, where nicotinamide could bereplaced with another amide (e.g. one of those discussed in Scheme 1)and where ICH₂ CH₂ OH could be replaced with another compound of thetype I--Z'--OH where Z' is C₁ -C₈ straight or branched alkylene.

Scheme 20 illustrates an alternate route to the derivatives of Scheme 8.This scheme represents a particularly attractive synthetic route to theprotected quaternary derivative 62. Moreover, the intermediate 176 canbe varied as discussed in conjunction with Scheme 19; also, this processcan be adapted to the preparation of derivatives of other--COOH--containing chelating agents, e.g. those of Schemes 1 and 2.

In Scheme 21, the intermediate 179, prepared as in Scheme 20, is used toprepare yet other compounds of the invention derived from3,4-diaminobenzoic acid.

Scheme 22 is illustrative of yet another variation in the procedure ofScheme 8. Scheme 22 can be readily adapted to the Preparation of otherderivatives of this invention; see, for example, the discussions ofSchemes 8 and 20 above.

In Scheme 23, there are depicted two highly desirable alternate routesto the quaternary salt 76 of Scheme 9. These alternate routes utilizethe quaternary activated esters of nicotinic acid to prepare thequaternary derivative 76 directly from the corresponding primary amine74. Use of either the succinimidyl or the phthalimidyl quaternaryintermediates (17 or 191) is illustrated. Other quaternary activatedesters for use in this reaction will be apparent from the variousprocesses described herein. After formation of the formula (I)quaternary such as 76, the process of Scheme 9 can then be used toprepare the other derivatives of this invention.

Scheme 24 depicts yet another highly desirable alternate route to thequaternary salt 76 of Scheme 9. In this particular preferred scheme, aprotecting group is introduced prior to introduction of the carrierfunction; the protecting group is then removed prior to reduction of thequaternary function to the corresponding dihydro. In the case of thechelating agent shown in this scheme, reaction with acetone Protectsboth the secondary amino and thiol functions by formation ofthiazolidine structures so that those functions do not interfere duringaddition of the carrier moiety. Subsequently, the secondary amino andmercapto groups are regenerated by reacting the protected intermediatewith mercuric chloride in an organic solvent such as methanol,conveniently at room temperature, and then decomposing the resultingcomplex with hydrogen sulfide. See, for example, British PatentSpecification No. 585,250, which utilizes such a procedure for theproduction of esters of penicillamine. After preparing the quaternarysalt 76 in this manner the process of Scheme 9 can be used to preparethe other derivatives of this invention. Variations in the procedureused, e.g. as discussed in connection with Scheme 23, can be used toobtain yet other derivatives of the invention.

Scheme 25 represents an alternate route to the compounds obtained viaScheme 14. The route uses the preferred route of introducing the carriermoiety in its quaternary form and can be readily adapted to thepreparation of derivatives of other --COOH containing chelating agentsand/or introduction of other carrier moieties disclosed herein. InScheme 26, there is illustrated a process for attaching a ##STR26##function or analogous carrier moiety to a pendant primary amine functionin a chelating agent. This process utilizes the thiazolidine structureto protect the secondary amino and thiol functions in the particularchelating agent depicted, as fully discussed in conjunction with Scheme24 above.

An alternate approach to the derivatives depicted in Scheme 26 is shownin Scheme 27, in which the primary amino group in the protected primaryamine is first converted to the corresponding --NHCO--R₃ --Br group,which is then reacted with nicotinamide or the like to afford theprotected quaternary intermediate.

Scheme 27 depicts a process for preparing carrier-containing derivativesof yet another type of chelating agent. The desired chelating agent inthis instance contains oxime functins, which are introduced after thequaternary form of the carrier has been attached. Formation ofderivatives of yet another type of chelating agent is depicted in Scheme29.

Similar schemes can be shown for the preparation of the otherderivatives of this invention. The steps of introducing and removingprotecting groups are only included when necessary. Also, the order ofsteps may be altered; in particular, quaternization may occur earlier inthe reaction scheme, depending of course on the particular compoundsinvolved. Other reaction schemes, reactants, solvents, reactionconditions etc. will be readily apparent to those skilled in the art.Also, insofar as concerns the quaternary derivatives, when an aniondifferent from that obtained is desired, the anion in the quaternarysalt may be subjected to anion exchange via an anion exchange resin or,more conveniently, by use of the method of Kaminski et al, Tetrahedron,Vol. 34, pp. 2857-2859 (1978). According to the Kaminski et al method, amethanolic solution of an HX acid will react with a quaternary ammoniumhalide to produce the methyl halide and the corresponding quaternary .Xsalt.

Reduction of the quaternary salt of formula (1) to the correspondingdihydro derivative of formula (II) can be conducted at a temperaturefrom about -10° C. to room temperature, for a period of time from about10 minutes to 2 hours, conveniently at atmospheric Pressure. Typically,a large excess of reducing agent is employed, e.g., a 1:5 molar ratio ofreducing agent to starting compound of formula (I). The process isconducted in the presence of a suitable reducing agent, preferably analkali metal dithionite such as sodium dithionite or an alkali metalborohydride such as sodium borohydride or lithium aluminum borohydride,in a suitable solvent. Sodium dithionite reduction is convenientlycarried out in an aqueous solution; the dihydro product of formula (II)is usually insoluble in water and thus can be readily separated from thereaction medium. In the case of sodium borohydride reduction, an organicreaction medium is employed, e.g., a lower alkanol such as methanol, anaqueous alkanol or other protic solvent. More typically, however, thequaternary of formula (I) is reduced in the same reaction mixture as thereduction of technetium to an appropriate oxidation state, affording theformula (III) radiopharmaceutical in one step from the formula (I)quaternary. Further details of the one-step reduction are givenhereinbelow.

It will be apparent from the foregoing that a wide variety ofderivatives of formulas (I) through (IV) can be obtained in accord withthis invention. In a particularly preferred embodiment of thisinvention, however, there are provided novel chelating agent precursorsof the formula ##STR27## wherein each R₅ is independently selected fromthe group consisting of H and C₁ -C₇ alkyl, or an R₅ can be combinedwith the adjacent >C--R₅ such that ##STR28## represents >C=O; each R₆ isindependently selected from the group consisting of H and C₁ -C₇ alkyl,or an R₆ can be combined with the adjacent >C--R₆ such that ##STR29##represents >C=O; ##STR30## is a radical of the formula ##STR31## whereineach R₇ is independently selected from the group consisting of H and C₁-C₇ alkyl; (alk) is a straight on branched lower alkylene group (C₁ -C₈)which additionally may contain 1, 2 or 3 oxygen atoms in the chain, saidoxygen atoms being nonadjacent to each other and also being nonadjacentto --A'--; X⁻ and n are as defined with formula (I); m' is a numberwhich when multiplied by n is equal to one; s is zero or one; --A--is--NH--, --COO--, --O--, --CONH--, ##STR32## wherein R₈ is C₁ -C₇ alkyl,or ##STR33## wherein R₉ is C₁ -C₇ alkyl; when --A--is --NH--, --O--or##STR34## then [QC⁺ ] is radical of any one of formulas (a) through (j)hereinabove; when --A-- is --CONH-- or ##STR35## has the imide structuredepicted above, then [QC⁺ ] is a radical of any one of formulas (k)through (s) hereinabove; and when --A-- is --COO--, then [QC⁺ ] is aradical of any one of formula (i) through (xiv) hereinabove. Preferablythe salts for formula (Ia) have the partial structure ##STR36## or areposition isomers and/or homologs of the first two partial structuresshown. It is also preferred that when ##STR37## then each R₇ ispreferably H and (alk) is preferably a C₁ -C₆ alkylene group, or a C₁-C₆ alkylene group interrupted by an oxygen atom in the chain; and thatwhen ##STR38## then (alk) is preferably a C₁ -C₆ alkylene group, or a C₁-C₆ alkylene group interrupted by an oxygen atom in the chain. When##STR39## is either of the above, then the presently preferred valuesfor --(alk)_(s) --A--are --COO--, --CH₂ O--, --CONH--, --CH₂ NH--and--CH₂ OCH₂ CH₂ O--, Preferred values for [QC+]in formula (Ia) are asgiven in conjunction with formula (I) hereinabove.

Corresponding to the preferred novel chelating agent precursors offormula (Ia) are the preferred novel chelating agents of the formula##STR40## wherein R₅ and R₆ are as defined with formula (Ia) and##STR41## is a radical of the formula wherein R₇, (alk), s and --A'--areas defined with formula (Ia); when --A'--is --NH--, --O--or ##STR42##wherein R₈ is C₁ -C₇ alkyl, then [DHC] is a radical of any one, offormulas (a') through (j'') hereinabove; when --A'--is --CONH--or##STR43## wherein R₉ is C₁ -C₇ alkyl or when ##STR44## has the imidestructure depicted above, then [DHC] is a radical of any one of formula(k') through (s'') hereinabove; and when --A'--is --COO--, then [DHC]is, a radical of any one of formulas (i') through (xiv'') hereinabove.Preferred compounds of formula (IIa) are then dihydro derivativescorresponding to the preferred compounds of formula (Ia).

Likewise preferred are the novel radiopharmaceuticals in which a formula(Ila) compound is chelated with a radioactive metal, especially withtechnetium. Especially preferred radiopharmaceuticals have the formula##STR45## wherein R₅ and R₆ are as defined with formula (Ia) and##STR46## is a radical of the formula ##STR47## wherein R₇, alk, s and--A'--are as defined with formula (Ia) and [DHC]is as defined withformula (IIa) above; and the corresponding quaternaries, "locked in" thebrain, especially those of technetium, which have the formula ##STR48##wherein R₅, R₆, m', X⁻ and n are as defined with formula (Ia) and##STR49## is a radical of the formula ##STR50## wherein R₇, alk, s,--A'--and [QC+] are as defined with formula (Ia) above. The preferredcomplexes of formulas (IIIa) and (IVa) are those which correspond to thepreferred derivatives of formulas (Ia) and (IIa).

Chelating agent precursors, chelating agents and radiopharmaceuticalswithin the purview of the present invention can also be prepared byreacting ##STR51## and thereafter reacting the amino group of theobtained compound 29 with the carboxyl group of compounds such as the2-oxopropionaldehyde bis (thiosemicarbazone) derivatives having a freecarboxyl group. Illustrative of such compounds is3-carboxy-2-oxopropionaldehyde bis(N-methylthiosemicarbazone), abifunctional chelating agent described in Yokoyama et al U.S. Patent No.4,287,362. The Yokoyama et al COOH--containing chelating agents also canbe derivatized as generally described hereinabove for derivatizing COOHgroups, e.g. as depicted in Schemes 1 and 2. Moreover, Yokoyama et al'schelating agents of the formula ##STR52## wherein R², R³ and R⁴ are eachH or C_(1-C) ₃ alkyl can be first converted to the corresponding esters(e.g. replacing --COOH with --COOC₂ H₅), which can then be reduced tothe corresponding alcohols (replacing --COOC₂ H₅ with --CH₂ OH) orconverted to the corresponding amides; the alcohols or amides can thenbe converted to the corresponding carrier-containing derivatives; see,for example, the discussion of Schemes 9-16 above. Other processvariations will be apparent from the many reaction schemes depictedhereinabove.

Another bifunctional chelating agent which can be readily converted tothe redox system-containing chelating agent precursors, chelating agentsand radiopharmaceuticals of this invention is a compound of the formula##STR53## which is also known as amino DTS and which is described in theliterature, e.g. in Jap. J. Nucl. Med. 19, 610 (1982). Amino DTS can bereadily converted to the derivatives of the present invention byreacting it with an activated ester of nicotinic acid or the like andquaternizing the resulting ester to afford the corresponding precursorof formula (I), which can then be utilized as generally described hereinto prepare the corresponding compound of formula (II) andradiopharmaceuticals of formulas (III) and (IV). See, for example,Scheme 30 below.

Yet another group of known chelating agents which is particularlywell-suited for conversion to the redox system-containing chelatingagent precursors, chelating agents and radiopharmaceuticals of thepresent invention can be represented by the formula ##STR54## whereinR¹, R², R³ and R⁴ are each H or C₁ -C₃ alkyl and n' is an integer of 0to 3. See, for example, Yokoyama et al U.S. Patent No. 4,511,550 andAustralian Patent No. 533,722. An especially preferred chelating agentencompassed by this group is known as amino-PTS, or AEPM, and has thestructure ##STR55## Amino-PTS can be converted to the derivatives of thepresent invention via the activated ester, as described supra inconnection with amino-DTS. See, for example, Scheme 33 below. The exactstructure of the resultant technetium complex 224 has not beendetermined; it is possible that the C=N and C=S bonds are also reducedduring one of the reduction steps. One possible structure for 224 is asfollows: ##STR56## (A similar structure could be depicted for complex165 of Scheme 30).

A preferred alternate route to derivatives of amino-PTS, amino-DTS andthe like is illustrated by Schemes 31 and 32 below. This route reacts aquaternized activated ester with the ligand containing a primary aminegroup to form the quaternary chelating agent precursor of formula (I) inone step. Variations in this highly desirable single step, as well as inthe two step alternative shown in Schemes 30 and 33 , will be apparentfrom the discussion of a number of reaction schemes depictedhereinabove. Also, it should be Pointed out that introduction of thecarrier moiety in its quaternary form, typically via a quaternizedactivated ester such as 191 or 17, is generally advantageous over thetwo step method, and any of Schemes 6-7 and 9-17 hereinabove could bereadily modified accordingly. ##STR57##

In a like manner, the presently contemplated carrier system can beincorporated into the structure of a novel technetium-99mradiopharmaceutical whose chelate portion is the residue of an amino-orhydroxy-substituted iminodiacetic acid, e.g.,N-[3-(1-naphthyloxy)2-hydroxypropyl] iminodiacetic acid. Suchsubstituted iminodiacetic acid chelating agents are known and aredescribed in Lobert et al. U.S. Pat. No. 4,017,596; such chelatingagents can be protected to the extent necessary and then thetrigonellinate or other carrier structure introduced through reactionwith the --NH₂ or --OH group in the chelating agent.

Similarly, suitable chelating agents and their precursors that include adihydropyridine⃡pyridinium salt carrier system can be prepared byreacting Compound 17 or the like with a chelating agent which is asubstituted-alkyl monophosphonic acid such as aminobutylphosphonic acid,1,5-diaminopentylphosphonic acid, and the like. Chelating agents of thisgeneral type are also known and are illustrated by those described inKohler et al. U.S. Pat. No. 3,976,762.

Yet other chelating agents containing one or two carboxyl functions aredescribed in Fritzberg U.S. Pat. No. 4,444,690. Carrier-containingtechnetium chelates corresponding to the Fritzberg chelates can beprepared as generally described hereinabove and as illustrated bySchemes 1 and 2 above.

Fritzberg U.S. Pat. No. 4,444,690 describes an interesting series of2,3-bis(mercaptoalkanoamide)alkanoic acid chelating agents of thegeneral formula ##STR58## wherein X is H or --COOH, and R and R' are Hor lower alkyl, and water-soluble salts thereof, used to prepare thecorresponding radiopharmaceuticals of the formula ##STR59## wherein X isH or --COOH, and R and R' are H or lower alkyl. The Fritzberg chelatingagents are prepared from the corresponding 2,3-diaminoalkanoic acids byesterification with a lower alkanol containing dry HCl, followed bytreating the resultant alkyl ester with a chloroalkanoyl chloride toform the bis(chloroalkanoamide)ester, followed by treating that esterwith ##STR60## followed by alkaline hydrolysis of the resultant2,3-bis(benzoylmercaptoalkanoamido)alkanoic acid ester to produce the2,3-bis(mercaptoalkanoamido)alkanoic acid chelating agent. Preparationof an analog from 3,4-diaminobenzoic acid is also disclosed byFritzberg. Many of Fritzberg's synthetic steps can be adapted to producethe formula (I) derivatives of this invention in which, in place of the--COOH group in Fritzberg's chelating agent, there is an (alk)_(s)--A'--[QC⁺ ] group wherein the structural variables are as defined withformula (Ia) hereinabove. See, for example, Schemes 12, 13 and 16 above.

Radiopharmaceuticals containing a dihydropyridine⃡pyridinium salt carriersystem can also be prepared using a novel chelating agent precursorobtained by reacting, in pyridine as the solvent, the aforementionedCompound 29 with nitrilotriacetic anhydride according to the knowngeneral procedure illustrated in Nunn et al U.S. Pat. No. 4,418,208.

The dicarboxyl pyridinium salt obtained from the above reaction isobtained in purified form as follows: The volatile components of thereaction mixture are evaporated to an oily semisolid on a rotaryevaporator. A solution of 10 percent aqueous sodium hydroxide (w/v) isused to dissolve the oily sem solid. The resulting solution is extractedwith methylene chloride to remove the remaining pyridine from theaqueous phase. The pH value of the aqueous phase is thereafter loweredto a value of about 6-8. The resulting aqueous solution is then reducedin me to about that of the original pyridine solution, and about fivetimes that volume of a saturated solution of picric acid is added toform a picrate derivative precipitate.

The picrate precipitate is washed with cold, distilled water, and isthen dissolved in a 10 percent aqueous solution of hydrochloric acid(v/v). The resulting solution is extracted with methyl chloride untilthere is no more yellow color in the aqueous or methylene chloridephases. The resulting, colorless aqueous phase is concentrated to aboutthe volume of the original pyridine solution, and is then lyophylized toprovide the chelating agent in dry form. The dried chelating agent isthen dissolved in ethanol and precipitated using the diethyl etherflooding technique described in Example 4 hereinbelow.

Still another useful chelating agent precursor can be prepared byreacting equimolar quantities of ethylenediaminetetracetic acid andacetic anhydride in dry pyridine following the teachings of Nunn et al.U.S. Pat. No. 4,418,208. and thereafter reacting a further equimolaramount of Compound (29) to form the monoamide adduct. The tridentatechelating agent salt is obtained as described immediately above.

The tridentate chelating agent precursor salt so obtained is thereafterreacted with the 99m pertechnate ion as described in Example 5 below,which reduces both the technetium and the pyridinium salt, to form a 1:1ligand:radioactive metal ion complex drug delivery system of thisinvention. The complex so formed is ionically neutral inasmuch as thefive valences of the reduced technetium-99m metal are taken up with oneoxygen atom and three carboxylate oxygens, and the pyridinium ring is inits reduced, dihydropyridine form.

As aforesaid, the preparation of the chelating agent precursors,chelating agents and radiopharmaceuticals of this invention must betailored to the particular starting materials used, especially asregards the presence of reactive functional groups in addition to thegroup which is to be linked to the carrier moiety. The stage at whichthe carrier is introduced and the manner in which the carrier isintroduced will be determined accordingly. Often the carrier must beintroduced in quaternary form at an early stage of the synthesis asillustrated hereinabove. When not so required, it may be more desirableto react an appropriate starting material such as nicotinic anhydridewith an NH₂ -- or OH-- containing ligand or ligand precursor, andquaternize at a later stage, after coupling the ligand (chelating agent)and the 3-pyridinecarbonyl group.

The processes depicted above are only intended to be illustrative. Manyvariations, for example, can be made in the chelating portions of themolecule, and such variations will naturally affect the syntheticscheme, particularly as regards the necessity for introducing protectinggroups and subsequent removal thereof.

In order to further illustrate the present invention and the advantagesthereof, the following specific examples are given, it being understoodthat same are intended only as illustrative and in no wise limitative.

EXAMPLE 1 N-(t-butoxycarbonyl),N-(2-mercaptoethyl)glycylN'-(2-aminoethyl)homocysteinamide (Compound 14 of Scheme 3)

N-(t-butoxycarbonyl),N-(2-mercaptoethyl)glycyl homocysteine thiolactone(13) is prepared as described in Examples 1 and 2 of Byrne et al U.S.Pat. No. 4,434,151, and is dissolved (1.0 gram; 3 millimoles) in 25milliliters of tetrahydrofuran (THF). The resulting solution is thencooled to about 0° C., and ethylenediamine (1.8 grams; 30 millimoles) isadded to form a new solution. The resulting new solution is maintainedfor about one hour. The volatile components of-the solution arethereafter removed with a rotary evaporator. n-Butanol (about 10milliliters) is added to the "dried" solution components and the liquidcomponents of the resulting composition are again removed by rotaryevaporation. The last step is repeated until the vapors remaining in theevaporation vessel do not cause a moistened pH-indicator paper toindicate a basic pH value, thereby also indicating that theethylenediamine has been substantially removed and that theN-(t-butoxycarbonyl),N-(2-mercaptoethyl)glycylN'-(2-aminoethyl)homocysteinamide so obtained is substantially pure.

EXAMPLE 2a Succinimidyl nicotinate (Compound 16 of Scheme 3)

Nicotinic acid (12.3 g; 0.1 mole) and N-hydroxysuccinimide (11.5 g; 0.1mole) are dissolved in 300 milliliters of hot dioxane. The mixture iscooled on an ice-bath and dicyclohexylcarbodiimide (20.6 g; 0.1 mole) in30 milliliters of dioxane is added. The reaction mixture is stirred,with cooling, for approximately three hours, then refrigerated for atleast 2 hours. The precipitated dicyclohexylurea is removed byfiltration, the solution is condensed under vacuum, and the yellowishsolids which precipitate are recrystalized from ethyl acetate. Whitecrystals (14 g) of succinimidyl nicotinate are obtained (yield 63.6%).Structure of the product is confirmed by NMR.

EXAMPLE 2b Succinimidyl Trigonellinate (Compound 17 of Scheme 3)

Succinimidyl nicotinate 16 (3.3 g; 15 mmole) is dissolved in 50milliliters of dioxane and 3 7 milliliters (8.2 g; 60 mmole) of methyliodide is added. The reaction mixture is reflexed for about 48 hours.The yellow crystals which precipitate during the reaction are removed byfiltration, washed with ethyl ether and dried under vacuum at 40° C.Succinimidyl trigonellinate (5.2 g) is obtained (Yield 96.3%). Structureof the product is confirmed by NMR.

An improved method for preparing Compound 17 is as follows:

A solution of 9.0 g (41 mmol) of the ester 16 and 11.6 g (82 mmol) ofmethyl iodide in 40 mL of anhydrous acetone is heated in a pressurebottle under an argon atmosphere for 16 hours. The yellow precipitatewhich forms is removed by filtration. YieId 14 g of Compound 17,darkening at 170° C. and melting at 197° C.

EXAMPLE 3 N-(t-butoxycarbonyl), N-(2-mercaptoethyl)glycylN'-[1-methyl-3-(2-N-ethyl)carbamoylpyridinium iodide]homocysteinamide(Compound 18 of Scheme 3)

N-(t-butoxycarbonyl), N-(2-mercaptoethyl)glycylN'-(2-aminoethyl)homocysteinamide--Compound 14--(1.12 grams; 0.003 mole)and succinimidyl trigonellinate--Compound 17--(0.70 gram; 0.0025 mole)are dissolved in 25 milliliters of dry pyridine with stirring. Anappropriately sized, "micro" Dean-Stark trap and condenser are added tothe reaction flask and the solution is heated to and maintained at atemperature of 80° C. until substantially all of the succinimidyl esteris replaced. The pyridine is removed on a rotary evaporator usingn-butanol as a "chaser" as described before for the ethylenediamineremoval. Once the pyridine is removed, the dried residue is trituratedwith THF and the solid is removed by filtration and washed several timeswith THF with care not to dry by air suction. The solid so obtained isthereafter dried in vacuo to provide Compound 18, N(t-butoxycarbonyl),N-(2-mercaptoethyl)glycyl N'-[1-methyl-3-(2-N-ethyl)carbamoylpyridiniumiodide]homocysteinamide.

EXAMPLE 4 N-(2-mercaptoethyl)glycyl N-[1-methyl-3-(2-N-ethyl)-carbamoylpyridinium iodide]homocysteinamide(Compound 19 of Scheme 3)

N-(t-butoxycarbonyl), N-(2-mercaptoethyl)glycylN'-[1-methyl-3-(2-N-ethyl)carbamoylpyridiniumiodide]homocysteinamide--Compound 18--(1.24 grams; 0.002 mole) isdissolved with stirring absolute ethanol (50 milliliters) and cooled toabout 0° C. in an ice-water bath HCl gas is bubbled through the stirredsolution for 15 minutes, and the solution is thereafter stirred for anadditional 15 minutes. Diethyl ether (200 milliliters) is thereafteradded to the solution to precipitate the salt. The precipitate isfiltered and washed with diethyl ether with care not to dry theprecipitate by air suction. The solid is then dried in vacuo to provideN-(2-mercaptoethyl)glycyl-N'-[1-methyl-3-(2-N-ethyl)carbamoylpyridiniumiodide]homocysteinamide.

EXAMPLE 5 Complex BetweenN-(2-mercaptoethyl)glycyl-N'-[1-methyl-3-(N-2-ethyl)carbamoyl-1,4-dihydropyridyl]homocysteinamideand the Oxotechnate-99m ion (Compound 20 of Scheme 3)

N-(2-mercaptoethyl)-glycyl-N'-[1-methyl-(2-N-ethyl)carbamoylpyridiniumiodide]homocysteinamide--Compound 19--(89 milligrams; 0.17 millimole) isdissolved in milliliter absolute ethanol and 1.0 milliliter of 1N NaOH.A 1.0 milliliter generator eluant of ^(99m) TcO₄ ⁻ (5 to 50 milliCuries)in saline is added. Then, 0.5 milliliter of dithionite solution,prepared by dissolving 336 milligrams of Na₂ S₂ O₄ per milliliter of 1.0NaOH, is added and the mixture heated sufficiently to reduce both thetechnetium and the pyridinium salt and to form the complex betweenN-(2-mercaptoethyl)glycyl-N'-[1-methyl-3-(N-ethyl)caramoyl-1,4-dihydropyridyl]homocysteinamideand the oxotechnate-99m ion. The complex so prepared is buffered by theaddition of 1.0 milliliter of 1N HCl and 4.0 milliliter of 0.1 molarNaH₂ PO₄, pH 4.5 buffer.

EXAMPLE 6 Complex BetweenN-(2-mercaptoethyl)glycyl-N'-[1-methyl-3-(N-2-ethyl)carbamoyl-1,4-dihydroquinolyl]homocysteinamideand the Oxotechnate-99 m ion

A radiopharmaceutical coupled to a carrier based upon a reduced,dihydroquinoline carrier such as the title complex can be preparedfollowing the steps outlined in Examples 1-5, but replacing thenicotinic acid in Example 2a with an equivalent quantity of3-quinolinecarboxylic acid.

EXAMPLE 7 N-[2-(acetamidomethyl)mercaptopropionyl]-glycylN'-(2-aminoethyl)homocysteinamide (Compound 25 of Scheme 4)

N-[2-(S-acetamidomethyl)mercaptopropionyl)glycyl homocysteinethiolactone (Compound 24 of Scheme 4), prepared as described in Examples7 and 9 of Byrne et al U.S. Pat. No, 4,434,151, is suspended (1.0 gram;3 millimoles) in 25 milliliters of THF. The resulting suspension iscooled to a temperature of about 0° C. In an ice-water bath, andethylenediamine (1.8 grams; 30 millimoles) is added to form a newsolution. N-[2-(acetamidomethyl) mercaptopropionyl]glycylN'-(2-aminoethyl)homocysteinamide is thereafter obtained in a mannersubstantially similar to that described in Example 1 for the analogouscompound.

EXAMPLE 8 N-(2-(acetamidomethyl)mercaptopropionyl]glycylN'-[1methyl-3-(2-N-ethyl)carbamoylpyridinium iodide-]homocysteinamide(Compound 26 of Scheme 4)

Compound 26 of synthetic Scheme 4 is obtained in a manner analogous tothat used in Example 3 to prepare Compound 19, but Compound 17 and 25are utilized as starting materials.

EXAMPLE 9 BetweenN-(2-mercaptopropionyl)glycyl-N'-[1-methyl-3-(2-N-ethyl)carbamoyl-1,4-dihydropyridine]homocysteinamideand the Oxotechnate-99m ion--(Compound 27 of Scheme 4)

Compound 26 of Example 8 (0.17 millimole) is dissolved in 1.0 milliliterof absolute ethanol and 1.0 milliliter of 1N NaOH. The complex of thisExample is thereafter prepared in a manner analogous to that describedfor the complex of Example 5. Here, the basic solution frees the2-mercaptopropionyl group from its protective N-methylene acetamidogroup while the dithionite reduces both the pyridinium and technetiumsalts.

EXAMPLE 10 3,4-dithia-2,2,5,5-tetramethylhexane-1,6-dione (Compound 68of Scheme 9)

To a stirred solution containing 115.6 g (1.6 mol) of isobutyraldehyde67 in 184 g of carbon tetrachloride are added dropwise, at 40°-50° C.,108 g (0.8 mol) of 97% sulfur monochloride. The addition is carried outduring a 2.5 hour period, under a nitrogen atmosphere, with occasionalcooling. The solution is maintained at 30°-45° C., with stirring, for anadditional 48 hour period under a current of nitrogen, to remove thehydrogen chloride liberated. The solution is distilled under vacuum togive 72 g of the desired 3,4-dithia-2,2,5,5-tetramethylhexane-1,6-dione,i.e. Compound 68 of Scheme 9. ¹ H NMR(CDCl₃) δ 9.1(s,2-CHO),1.4[s,12,C(CH₃)₂ -].

EXAMPLE 11 Ethyl 2,3-(diammonium)propionate dichloride (Compound 70 ofScheme 9)

To 10 g (0.07 moll of ethyl cyanoglyoxylate-2-oxime 69 are added 125 mLof absolute ethanol, 15 g of hydrogen chloride gas and 1 g of platinumoxide. The mixture is hydrogenated using a Parr-hydrogenation apparatus.Hydrogen uptake is complete in 3 hours. The product is removed byfiltration and taken up in 75 mL of hot 95% ethanol. The ethanolsolution is filtered. The filtrate is then cooled and the crystallineproduct which separates on standing is removed by filtration. There isthus obtained ethyl 2,3-(diammonium)propionate dichloride, i.e. Compound70 of Scheme 9. Yield 5 g (35%), melting point 164°-66° C. (lit.164.5°-165° C.); ¹ H NMR(D₂ O) δ 4.5(m,3,--NCHCO--, --OCH₂ CH₃),3.5(m,2,--NCH₂ CH--), 1.3(t,3,--OCH₂ CH₃).

EXAMPLE 125,8-diaza-1,2-dithia-6-ethoxycarbonyl-3,3,10,10-tetramethylcyclodeca-4,8-diene(Compound 71 of Scheme 9) Procedure I

To 1.0 g (5 mmol) of the bisaldehyde 68 is added dropwise a solution of1.0 g (5 mmol) of the ester 70 and 0.9 mL of pyridine in 30 mL ofmethanol at 0° C. while under a nitrogen atmosphere. The addition takesplace during a 10 minute period. The solution is then allowed stand for1 hour, after which time 10 mL of water is added. The solution turnsturbid and warms to 26° C. The solution is stirred for an additional 20minute period, after which time the white precipitate which formssettles out of solution. The precipitate is removed by filtration andthen taken up in chloroform. The chloroform solution is dried oversodium sulfate. Removal of the solvent and trituration of the residuewith petroleum ether gives white plate-like crystals of the desiredproduct,5,8-diaza-1,2-dithia-6-ethoxycarbonyl-3,3,10,10-tetramethylcyclodeca-4,8-diene,i.e. Compound 71 of Scheme 9, in 53% yield (1 g), melting point 98°-99°C. IR (thin film) 3450, 1740, 1650 cm⁻¹ ; ¹ H NMR(CDCl₃) δ6.9(m,2,C-N═CH--), 3.0-4.6(m,5,--OCH₂ CH₃, --NCH₂ CH--N--),1.5[m,15,2>C(CH₃)₂, --OCH₂ CH₃ ].

Procedure II

To 1.0 g (5 mmol) of the bisaldehyde 68 in 10 mL of methanol is addeddropwise 1.0 g (5 mm of the ester 70 and 1 g (12 mmol) of sodiumbicarbonate in 20 mL of a 50:50 by volume mixture of methanol and water.The mixture is stirred at 0° C. for 10 minutes, after which time 10 mLof water is added. The resultant mixture is maintained at roomtemperature, with stirring, for 2 hours. Water is added until the whiteprecipitate which forms separates out of solution. The precipitate isremoved by filtration and taken up in chloroform. Removal of the solventby rotary evaporation affords 0.4 g (21% yield) of Compound 71, having amelting point and ¹ H NMR spectrum identical the product of Procedure I.

Procedure III

A solution of 8 g of the ester 70 and 7 mL of pyridine in 200 mL ofmethanol is added dropwise over a two hour period to a solution of 8 gof bisaldehyde 68 in 25 mL of methanol. The reaction mixture is cooledin an ice bath after the addition for 1 hour, then is allowed to remainat room temperature for 1 hour. The reaction mixture is then placed in afreezer (-20° C.) overnight. The solution is concentrated to one-thirdvolume water is added and the aqueous solution is extracted withchloroform. The chloroform extract is washed with saturated aqueoussodium chloride solution and dried over magnesium sulfate. Removal ofthe solvent leaves a viscous mass, which is dissolved in 20 mL ofhexane. The hexane solution is cooled in an acetone/dry ice bath until awhite powder separates. The product is removed by filtration and takenup in chloroform The chloroform solution is concentrated. White crystalsof Compound 71 are formed on standing. Yield 7 g, melting point 95°-96°C. NMR and IR as in Procedure I.

EXAMPLE 13 6-carbamoyl-5,8-diaza-1,2-dithia-3,3,10,10-tetramethylcyclodeca-4,8-diene (Compound 72of Scheme 9) Procedure I

A solution of 5 g of the ester 71 in 20 mL of tetrahydrofuran and 20 mLof aqueous ammonia is stirred at room temperature for 2 hours, afterwhich time it is allowed to stand at room temperature for 24 hours.Removal of solvent leaves a white powder which is removed by filtration.The product,6-carbamoyl-5,8-diaza-1,2-dithia-3,3,10,10-tetramethylcyclodeca-4,8-diene,i.e. Compound 72 of Scheme 9, is crystallized from a mixture ofisopropanol and water. Yield 4 (88%), melting point 181°-183° C. IR(KBr) 3300, 3100, 1650 cm⁻¹ ; ¹ H NMR(CDCl₃) δ7.0(m,2,--HC═N--),6.4(broad band,2, --CONH₂), 3,8-4.6[m,3, --NCH₂ --CH(N--)CO--], 1.5,1.4[s,12,>C(CH₃)₂ ].

Procedure II

A solution of 5 g of the ester 71 in 20 mL of tetrahydrofuran, 20 mL ofethanol and 20 mL of aqueous ammonia (28%) is stirred at roomtemperature for 16 hours. Removal of the solvent leaves Compound 72 as awhite powder, which crystallizes from toluene as white plates. Yield 4g, melting point 193°-194° C. IR and NMR as in Procedure I.

EXAMPLE 145-carbamoyl-5,8-diaza-1,2-dithia-3,3,10,10-tetramethylcyclodecane(Compound 73 of Scheme 9)

To 3.7 g of the amide 72 in 25 mL of 95% ethanol is added 2 g of sodiumborohydride. The mixture is stirred at room temperature for 2 hours,then is heated at reflux for 2 hours. The solution is thereafterconcentrated in vacuo and water is added to precipitate the product. Thewhite crystalline product is removed by filtration. Recrystallizationfrom a mixture of isopropanol and water affords6-carbamoyl-5,8-diaza-1,2-dithia--3,3,10,10-tetramethylcyclodecane, i.e.Compound 73 of Scheme 9, as fine white needles melting at 138°-139° C.Yield 3 g. ¹ NMR(CDCL₃) δ 2.3-4.0[m,7, --NCH,CH--N--, 2--NCH₂--C(CH₃)--S--], 1.8(broad band, 2, --CONH₂), 1.3[m,14, C(CH₃)₂,--CNH--CH₂ --].

EXAMPLE 15 5-aminomethyl-4,7-diaza-2,9-dimethyldecane-2,9-dithiol(Compound 74 of Scheme 9)

A solution of 1.8 g of the amide 73 in 50 mL of dry tetrahydrofuran isadded dropwise to a slurry of 1 g of lithium aluminum hydride in 100 mLof dry tetrahydrofuran. The addition takes place over a 30 minute periodThe mixture is then heated at the reflux temperature for 20 hours. Atthe end of that time, the reaction mixture is first cooled and thenquenched with saturated Na-K tartrate solution. The aqueous phase isextracted with chloroform. The combined organic phase is then dried oversodium sulfate. Removal of the solvent by rotary evaporation affords, asa viscous oil, 5-aminomethyl-4,7-diaza-2 9-dimethyldecane-2,9-dithiol,i.e. Compound 74 of Scheme 9; ¹ H NMR (CDCl₃) δ2.8[m,9,--NCH₂CH--C(CH₂)NH--, 2--NCH₂ --C(CH₃)₂ S--], 1.5[m,14,>C(CH₂, --SH].

EXAMPLE 16 5,8-diaza-1,2-dithia-3,3,10,10-tetramethylcyclodeca-4,8-diene(Compound 87 of Scheme 11)

To 3.15 g of the dialdehyde 68 is added 4.0 g of ethylenediamine, withstirring and cooling, over a period of 10 minutes. The thick mass whichresults is stirred for an additional one minute period, then allowed tostand for 1 hour at room temperature and subsequently cooled for 16hours in a freezer (-20° C.). The solid is removed by filtration andwashed with 500 mL of water. The white product is then taken up inchloroform and the chloroform solution is dried over sodium sulfate.Removal of the chloroform gives 2.5 g of5,8-diaza-1,2-dithia-3,3,10,10-tetramethylcyclodeca-4,8-diene, i.eCompound 87 of Scheme 11, as a white crystalline product, melting at168°-170° C. (lit. 162°-164° C. 163°-166° C.) ¹ H NMR(CDCl₃) δ6.9(s,2,--HC═N--), 4 2,3.0(doublet of doublet, 2, 2--CH₂ --CH.sub. 2),1.40[s,6,--C(CH₃)₂ ]. Anal. Calcd. for C₁₀ C₁₈ N₂ S₂ : C, 52.13; H,7.88; N, 12.16; S, 27.83. Found: C, 52.20; H, 7.90; N, 12.14; S, 27.74.

EXAMPLE 17 5,8-diaza-1,2-dithia-3,3,10,10-tetramethylcyclodecane(Compound 88 of Scheme 11)

A solution of 0.5 g of 87 and 0.3 g of sodium borohydride in 23 mL ofethanol is stirred at room temperature for 1 hour, then is heated at thereflux temperature for 20 minutes. Then, 10 mL of water are added andthe mixture is heated for an additional 10 minutes. The solvent ispartially removed by rotary evaporation and the residue is extractedthree times with 10 mL portions of chloroform. The chloroform extract isdried over sodium sulfate and the solvent is removed by rotaryevaporation. The resultant liquid solidifies on cooling. Flashchromatography (eluent hexanes/dichloromethane/isopropanol 5:1:1 byvolume) gives 5,8-diaza-1,2-dithia-3,3,10,10-tetramethylcyclodecane,i.e. Compound 88 of Scheme 11, as a solid, melting at 52°-53° C. ¹ HNMR(CDCl₃) δ 3-2.1(m,10 ring protons), 1.1,1 2(s,6 CH₃, CH₃).

EXAMPLE 18N-[(4,7-diaza-2,9-dimercapto-2,9-dimethyldec-5-yl)methyl]nicotinamide(Compound 75 of Scheme 9)

A solution of 9 mmol of the activated ester 16 in 30 mL ofdimethoxyethane is added dropwise over a period of one hour to 8.4 mmolof the amine 74 in 70 mL of dimethoxyethane. Thin layer chromatographyafter one hour, using a solvent system of petroleumether/acetone/dichloromethane/isopropyl alcohol (10:5:5:1 by volume),indicates a major component has been obtained. The solvent is removed byevaporation and the residue is treated with water. The resultant mixtureis extracted with chloroform and dried over sodium sulfate. Removal ofthe solvent affords the desired product, Compound 75 of Scheme 9.

EXAMPLE 193-{N-[(4',7'-diaza-2',9'-dimercapto-2',9'-dimethyldec-5'-yl)methyl]carbamoyl}-1-methylpyridiniumiodide (Compound 76 of Scheme 9)

Compound 75 is reacted with methyl iodide according to the generalprocedure described in Example 2b above. Prepared in this manner is thedesired quaternary salt, i.e. Compound 76 of Scheme 9.

EXAMPLE 20 Complex formation

The general procedure of Example 5 can be repeated to convert the otherquaternary salts of formula (I) to the correspondingradiopharmaceuticals, e.g. to convert Compound 76 to Complex 78,Compound 83 to Complex 85 and so forth.

EXAMPLE 21 5-aminomethyl-4,7-diaza-2,9-dimethyldecane-2,9-dithiol(Compound 74 of Scheme 9)

To a slurry of 11 g of lithium aluminum hydride in 300 mL of drytetrahydrofuran is added dropwise, over a 2 hour period and under anargon atmosphere, 13 g of the amide 72 in 150 mL of dry tetrahydrofuran.After the addition is complete, the reaction mixture is heated at refluxfor 30 hours, then quenched with saturated Na-K tartrate solution.Treatment with 3N hydrochloric acid and then with saturated sodiumcarbonate solution, followed by fitration and extraction of the filtratewith dichloromethane, affords an organic solution which is dried overmagnesium sulfate. Removal of the solvent affords the desired amine,Compound 74 of Scheme 9, as a viscous oil.

A sample of the free amine thus obtained is dissolved in diethyl etherand hydrogen chloride gas is added. The white powder which separates isremoved by filtration and purified from ethanol/water to give thecorresponding hydrochloride salt melting at 225°-228° C. ¹ H NMR (D₂ O)δ 3.3-4.2(m,9H,HCl,NH₂ CHH₂, --HCl NHCH₂), 1.5[m,12H,C(CH₂)₂ ]. Anal.Calcd. for C₁₁ H₃₀ Cl₃ N₃ S₂. H₂ O: C,33.63; H,8.21; N,10.69; Cl,27.07;S,16.32. Found: C,33.93; H,7.94; N,10.60; Cl,27.05; S,16.25.

EXAMPLE 22 Compound 192 of Scheme 24

A mixture of 1 g of the amine 74, 75 mL of acetone and a catalyticamount of p-toluenesulfonic acid is heated at reflux for 24 hours. Thesolvent is removed by rotary evaporation and the residue is taken up inchloroform and treated successively with saturated aqueous sodiumbicarbonate solution, aqueous sodium hydroxide solution (10%) andsaturated aqueous sodium chloride solution. The solution is dried overmagnesium sulfate. Removal of the solvent leaves a viscous mass. Thinlayer chromatography (CHCl₃ /methanol, 2:1) indicates two majorcomponents having R_(f) values of 0.13 and 0.73. The component with thelower R_(f) value shows a positive ninhydrin test, confirming that it isthe desired primary amine 192, while the component with the higher R_(f)value is negative. ¹ H NMR of the R_(f) 0.73 component (CDCl₃): δ 2.9,2.5, 1.3-1.5. ¹ H NMR of the R_(f) 0.13 component (CDCl₃): δ 3.0, 2.8,2.3, 1.2-1.7. Obtained in this manner is the desired bisthiazolidineprimary amine, Compound 192 of Scheme 24.

EXAMPLE 23 Compounds 193 and 76 of Scheme 24

Reaction of the bisthiazolidine primary amine 192 with the quaternizedactivated ester 17 or 191 affords the corresponding bisthiazolidinequaternary, i.e. Compound 193 of Scheme 24, which can then bede-protected, e.g. by reaction with mercuric chloride, followed bytreatment with hydrogen sulfide, to give the unprotected quaternary,Compound 76 of Scheme 24.

EXAMPLE 24 Compound 81 of Scheme 10

A solution of 7 g (3 mmol) of the ester 71 in 50 mL of drytetrahydrofuran is added dropwise over a period of 1 hour to 1.8 g (47mmol) of lithium aluminum hydride in 200 mL of dry tetrahydrofuran. Themixture is heated at reflux for 16 hours, after which time the reactionis quenched with K-Na tartrate solution. The organic phase is dried oversodium sulfate. Removal of the solvent leaves a yellow viscous mass.Yield 4 g (65%) of the desired alcohol, Compound of Scheme 10. ¹ H NMR(CDCl₃) δ 2.2-2.8, 3.5, 2. 1.5.

EXAMPLE 25 Compound 83 of Scheme 10

Following the general procedure of Example 22, but substituting anequivalent quantity of the alcohol in place of the amine 74, affords thebisthiazolidine alcohol, i.e. the protected counterpart of Compound 81of Scheme 10. That protected alcohol can then be subjected to theprocedures detailed in Example 23 above to ultimately give thecorresponding unprotected quaternary, Compound 83 of Scheme 10.

EXAMPLE 26 Compound 32 of Scheme 5

A solution of 17 mL of 2N lithium borohydride in tetrahydrofuran isadded to 300 mL of dry tetrahydrofuran under an argon atmosphere. Tothat solution are added 10 g (0.035 mol) of the ester 40 in 100 mL ofdry tetrahydrofuran. The resultant cloudy solution is heated at refluxfor 1.5 hours. The reaction is quenched with water and the organic phaseis washed with saturated aqueous sodium chloride solution and dried overmagnesium sulfate. Removal of the solvent leaves, as a white powderwhich is very soluble in water, the corresponding primary alcohol,Compound 32 of Scheme 5. Yield 2 g (24%); melting point 85°-90° C.; ¹ HNMR (acetone-d₆) δ 7-8, 4.15, 3.3-4.0.

EXAMPLE 27 Compound 33 of Scheme 5

To 1 g of the alcohol 32 in 40 mL of dry ethanol is added a solution ofsodium thiobenzoate prepared from 0.2 g of sodium in 10 mL of ethanoland 1.26 g of thiobenzoic acid in 5 mL of ethanol. The reaction mixtureis stirred at room temperature for 10 minutes, then is heated at 45° C.for an additional 10 minutes. The mixture becomes very thick anddifficult to stir and a yellow product separates. The product, Compound33 of Scheme 5, is removed by filtration and washed with water. Yield1.2 g, melting point 151°-152° C., ¹ H NMR (DMSO-d₆ /acetone-d₆) δ7.4-8.3, 3.85, 3.1-3.6.

Example 28 Compound 168 of Scheme 18

Cyanoacetic acid (8.5 g; 0.1 mol) and N-hydroxysuccinimide (11.5 g; 0.1mol) are combined in 150 mL of dry tetrahydrofuran. To the cooledsuspension is added dropwise a solution of 20.6 g (0.1 mol) ofdicyclohexylcarbodiimide in 50 mL of dry tetrahydrofuran over a periodof 2 hours. The mixture is allowed to warm to room temperatureovernight. The white precipitate which forms is removed by filtrationand washed with 50 mL of tetrahydrofuran. The combined filtrates areconcentrated to give 8 g (44% yield) of the ester 167. The productcrystallizes from isopropyl alcohol as white needles m.p. 140°-142° C.

A solution of the ester 167 (0.62 g, 3.4 mmol) in 10 mL of drydimethoxyethane added dropwise to a stirred solution of the cyclicdiamine 88 (0.8 g, 3.4 mmol) in 20 mL of dry dimethoxyethane at roomtemperature. The solution is stirred for an additional 2 hours, afterwhich it is allowed to stand for 16 hours. The dimethoxyethane isremoved by rotary evaporation and the brown residue is suspended inwater to remove N-hydroxysuccinimide. The product 168 is removed byfiltration and crystallized from toluene/hexanes as fine brown needles;yield 0.9 g (88%); m.p. 142°-143° C.; IR (thin film) 3450, 2250, 1675cm⁻¹ ; ¹ HNMR (CDCl₃): δ 3.7, 2.4-3.6, 3.5, 1.3, 1.25. Anal. Calcd. forC₁₃ H₂₃ N₃₀ S₂ : C, 51.79; H, 7.69; N, 13.94; S, 21.27. Found: C, 51.99;H, 7.12; N, 14.01; S, 21.34.

EXAMPLE 29 Compound 169 of Scheme 18

A solution of 3 g of the nitrile 168 in 50 mL of dry tetrahydrofuran isadded dropwise over a 30 minute period to a stirred slurry of 1.2 g oflithium aluminum hydride in 100 mL of dry tetrahydrofuran, under anitrogen atmosphere. The pale yellow solution is heated at reflux for 7hours, then stirred at room temperature for 50 hours. The slurry ishydrolysed with a saturated Na-K tartrate solution, the aqueous phase isextracted with dichloromethane and the combined organic extracts aredried over sodium sulfate. Rotary evaporation of the solution leaves theamine 169 as a viscous yellow oil.

EXAMPLE 30 Compound 170 of Scheme 18

A solution of the activated ester 16 (2 g, 9 mmol) in 30 mL ofdimethoxyethane is added dropwise over a period of 1 hour to the amine169 (2.6 g, 8.9 mmol) in 70 mL of dimethoxyethane. After 1 hour, thinlayer chromotography (eluent: petroleumether/acetone/dichloromethane/isopropyl alcohol, 10:5:5:1) indicates onemajor component having an R_(f) of 0.6. The solvent is removed byevaporation, the residue is treated with water and the mixture isextracted with chloroform and dried over sodium sulfate. Removal of thesolvent leaves 170 as a viscous yellow mass, yield 2.1 9; ¹ HNMR (CDCl₃)δ 7.3-9.3, 2.6-3.6, 1.5.

EXAMPLE 31 Compound 40 of Scheme 19

To a mixture of 10 g of sodium bicarbonate in 50 mL of water and 200 mLof toluene is added the ester 70 (2 g, 0.01 mol), with cooling in anice-bath. Chloroacetyl chloride (5 g, 0.44 mol) solution is addeddropwise, then the mixture is allowed to warm to room temperature. Theorganic phase is extracted with ethyl acetate, washed with water andbrine and then dried over magnesium sulfate. Removal of the solventleaves as a white mass; yield 2 g (70%); m.p. 85°-87° C. ¹ HNMR (CDCl₃):δ 7.12, 7.6, 4.67, 4.2, 4.07, 3.75, 1.3.

EXAMPLE 32 Compound 41 of Scheme 19

A solution of the ester 40 (2 g, 9 mmol) in 20 mL of dry ethanol isprepared under argon. To this is added a solution of sodium thiobenzoatein dry ethanol (prepared from 0.45 g of Na in 20 mL of ethanol to formsodium ethoxide, which is reacted with 2.5 g of 97% thiobenzoic acid).Precipitation occurs immediately. The reaction mixture is heated atreflux for 5 minutes, then is diluted with ethyl acetate. The aqueousphase is extracted with ethyl acetate The combined organic extracts arewashed with water and brine and dried over magnesium solvent. Removal ofthe solvent leaves 4.1 g of a creamy white powder. Crystallization fromtoluene gives 2.4 g of white product, 41, m.p. 125°-127° C. (Lit.29.5°-131° C.). NMR is consistent with structure.

EXAMPLE 33 Compound 176 of Scheme 19

A mixture of iodoethanol (7.5 g, 43 mmol), nicotinamide (5.2 g, 43 mmol)and 150 mL of acetone is heated at reflux for 18 hours. The mixture iscooled and the product 176 is removed by filtration. Yield 1.5 g (12%);m.p. 87° C.

EXAMPLE 34 Compound 177 of Scheme 20

To a mixture of 10 g of potassium carbonate in 20 mL of water and 200 mLof toluene is added 5 g(32 mmol) of 3,4-diaminobenzoic acid. To thecooled mixture is added 14.4 g (127 mmol) of chloroacetyl chloride in 10mL of toluene over a period of 1 hour. After the addition is complete,the mixture is stirred at room temperature for 30 minutes. The brownproduct is removed by filtration and crystallized from ethanol. Yield 8g(82%) of 177, m.p. 240°-241° C.

EXAMPLE 35 Compound 178 of Scheme 20

To 25 mL of ethanol is added 0.17 g of sodium metal, followed by 1.1 g(7.4 mmol) of thiobenzoic acid. To the resultant yellow-brown solutionis added 1.16 g (3.7 mmol) of the acid 177. The mixture turns yellowimmediately and thickens. The mixture is diluted to 200 mL with dryethanol and heated at reflux for 2 hours. The product is removed byfiltration and crystallized from isopropyl alcohol/tetrahydrofuran.Yield 1 g (53%) of 178, m.p. 244°-245° C.

EXAMPLE 36 Compound 179 of Scheme 20

To 15.2 g (0.03 mol) of the acid 178 and 3.45 g (0.03 mol) ofN-hydroxysuccinimide is added 500 mL of dry tetrahydrofuran. To theresultant suspension is added 6 g (0.03 mol) of dicyclohexylcarbodiimidein 50 mL of dry tetrahydrofuran, over a period of 1 hour. The resultingmixture is stirred at room temperatre for 16 hours. The whiteprecipitate of dicyclohexylurea which forms is removed by filtration andthe filtrate in concentrated in vacuo to give a brown product. Flashchromatography of a small sample (eluent: dichloromethane/aetone, 3:1)gives the ester 179, m.p. 117°-118° C.

EXAMPLE 37 Compound 183 of Scheme 21

A solution of 2-bromoethylamine hydrobromide (10.2 g, 0.05 mol) andnicotinamide (6 g, 0.05 mol) in 150 mL of dry dimethylformamide isheated at 140° C. for 16 hours. The precipitate which forms is removedby filtration and washed with ether. Yield 14 g (88%), m.p. 280° C.(decomp.) of 183.

EXAMPLE 38 Compound 75 of Scheme 9

A solution of the amine 74 (2 g, 7.5 mmol) and the activated ester 16(1.65 9, 7.5 mmol) in 100 mL of dry dimethoxyethane is stirred at roomtemperature for 24 hours. The solvent is removed by rotary evaporationand the residue is treated with water. The viscous product is extractedwith chloroform and dried over magnesium sulfate. Removal of the solventleaves 75 as a viscous mass. NMR is consistent with structure. Thecompound is used without further purification.

EXAMPLE 39 Compound 76 of Scheme 9

A solution of the amid 75 (0.5 g), 5 mL of methyl iodide and 20 mL ofnitromethane is stirred at room temperature for 7 days under argon.After the second day, a precipitate begins to form. The precipitate isremoved by filtration and treated with acetone. Yield 150 mg of thequaternary salt m.p. 210°-215° C. (decomp.) ¹ H NMR (DMSO-d₆) δ 8.3-9.5,4.5, 3.0-4.0, 1.2-1.5.

EXAMPLE 40 Compound 77 of Scheme 9

To a solution of 0.5 g (1 mmol) of the quaternary salt 76 in 10 mL ofwater is added 0.25 g (3 mmol) of sodium bicarbonate and 0.61 g (3 mmol)of sodium dithionite. Ether (50 mL) is added and the mixture is stirredunder a nitrogen atmosphere for 30 minutes while being cooled in anice-water bath. The aqueous phase is extracted with dichloromethane. Thecombined organic phase is dried over magnesium sulfate. Obtained in thismanner is the dihydro derivative 77.

EXAMPLE 41 Compounds 81 and 81a of Scheme 10

A solution of the ester 71 (10 g, 35 mmol) in 100 mL of drytetrahydrofuran is added dropwise over a period of 30 minutes to aslurry of lithium aluminum hydride (4 g, 94 mmol) in 300 mL of drytetrahydrofuran, with cooling in an ice-bath. The slurry is then heatedat reflux for 24 hours. The reaction is quenched with saturated Na-Ktartrate solution, then with 3N hydrochloric acid and finally withsodium carbonate. The aqueous phase is extracted with chloroform. Thecombined organic phase is washed with saturated aqueous sodium chloridesolution and dried over magnesium sulfate. Removal of the solvent leavesthe alcohol 81 as a viscous mass. The product is taken up in ethersaturated with hydrogen chloride, with cooling in an ice-bath. Yield 6 gof the salt 81a, m.p. 190°-191° C. NMR and elemental analysis consistentwith structure.

EXAMPLE 42 Compound 190 of Scheme 23

To 24.6 g (0.17 mol) of nicotinic acid and 32 g (0.19 mol) ofN-hydroxyphthalimide in 300 mL of tetrahydrofuran are added 41 g ofdicyclohexylcarbodiimide in 200 mL of tetrahydrofuran voer a period of 2hours. The reaction mixture is stirred at room temperature for 24 hours.The white precipitate of dicyclohexylurea which forms is removed byfiltration. The filtrate is concentrated, leaving a white mass which iscrystalllized twice from ethyl acetate, once from isopropylalcohol, andagain from ethyl acetate. The various batches of 190 thus obtained meltat 132°-135° C. and 148°-150° C. ¹ H NMR (CDCl₃) δ 8.4-9.5 (m, 3H,Py-H); 7.95 (s, 4H, Ar-H); 7.5-7.7 (m, 1H, Py-H).

EXAMPLE 43 Compound 191 of Scheme 23

A solution of the ester 190 (5 g, 18.6 mmol) and methyl iodide (6 g,42.4 mmol) in 40 mL of acetone is placed in a pressure bottle and heatedin an oil bath (bath temperature 65° C.) for 12 hours. The product isremoved by filtration. Yield 4.5 g (59%) of the quaternized activatedester 191. The product darkens at 175° C. and melts at 185° C. ¹ HNMR(DMSO-d₆) δ 8.2-9.9 (m, 4H, Py-H); 8.1 (s, 4H, Ar-H); 4.52 (s, 3H,N-CH₃).

EXAMPLE 44 Compound 180 of Scheme 21

To the activated ester 179 (9 g, 14.9 mmol) in 100 mL of dimethoxyethaneis added ethanolamine (0.918 g, 15.4 mmol) in 50 mL of dimethoxyethane.The reaction mixture is stirred at room temperature for 48 hours; thewhite precipitate which forms is then removed by filtration.Concentration of the solvent gives an additional 2 g of the product.Yield 4 g (49%) of 180, melting at 205°-210° C. ¹ HNMR (DMSO-d₆): δ7.5-10, 4.8, 3.3-3.7, 3.3.

EXAMPLE 45 Compound 179 of Scheme 25

The acid 178 (8 g) and N-hydroxysuccinimide (1.8 g) are combined in 200mL of tetrahydrofuran. To that suspension is addeddicyclohexylcarbodiimide (3.16 g) in 25 mL of tetrahydrofuran over aperiod of 2 hours. The mixture is then stirred at room temperature for16 hours. The white precipitate is removed by filtration and thefiltrate is concentrated in vacuo. The product, the activated ester 179,is crystallized from toluene.

EXAMPLE 46 Compound 194 of Scheme 25

To 4.7 g (8 mmol) of the activated ester 179 is added a solution of 0.14g (8 mmol) of ammonia in 150 mL of dimethoxymethane. The reactionmixture is stirred at room temperature for 16 hours. The solution isconcentrated in vacuo to give 3 g of the amide 194 as a white product.

EXAMPLE 47 Compound 76 of Scheme 23

To 6.12 g (23 mmol) of the amine 74 in 100 mL of anhydrousdimethylformamide is added dropwise, over a period of 4 hours, 2.2 g (6mmol) of the activated ester 17 in 80 mL of anhydrous dimethylformamide.The reaction is carried out at -47° C. (acetonitrile/dry ice) whileunder an argon atmosphere. The reaction mixture is stirred for anadditional 2 hours at -47° C., then is placed in a freezer(approximately -20° C.) overnight. The dimethylformamide is removed invacuo. To the residue is added 150 mL of xylene and the solvent is againremoved in vacuo. The residue is taken up in 75 mL of benzene andtriturated with petroleum ether, after which a gummy product separates.This process is repeated twice. The resultant gummy residue is suspendedin the same solvent. HPLC data indicate one major peak with some aminebeing present. Flash chromatography (eluent, methanol) of a small sampleof the reaction mixture gives a product which by HPLC indicates twocomponents, the residual amine and the desired quaternary salt 76.

EXAMPLE 48 Compound 76 of Scheme 23

To 1.5 g (5.7 mmol) of the amine 74 in 20 mL of dimethylformamide isadded 0.5 g (1.4 mmol) of the quaternary compound 191 in 20 mL ofdimethylformamide. The reaction is carried out at -47° C. over a twohour period, under an argon atmosphere. The solvent is removed in vacuoand the residue is treated 5 times with benzene/petroleum ether. HPLCdata again indicates most of the amine has been removed, leaving thedesired quaternary salt 76.

EXAMPLE 491-methyl-3-[N-{{β-{4-[1',2'-bis(4"-methylthiosemicarbazono)prop-1'-yl]phenyl}ethyl}}carbamoyl]pyridiniumiodide hemihydrate (Compound 222 of Scheme 32)

Amino-PTS hydrochloride monohydrate (100 mg, 0.238 mmol) in dry pyridine(15 mL) with the quaternized activated ester 192 (200 mg, 0.488 mmol) isheated at entle reflux. After 2 hours, no amino-PTS remains and themixture is set aside to cool. Volatiles are removed in vacuo and theresidue is washed with water (10 mL) and taken into chloroform (40 mL).The aqueous layer is re-extracted with chloroform (20 mL) and thecombined, dried (MgSO₄) organic layers are evaporated dryness, leavingan orange oil. The oil is taken into a minimum of warm ethanol.Trituration results in precipitation of a pale yellow powder. Yield 75mg (51%) of the quaternary salt 222, melting at 214°-216° C. IR (KBr)3000-3600, 1670, 1535, 1470 cm⁻¹ ; ¹ HNMR (DMSO-d₆) δ 9.5, 8.1-9.4,7.1-7.6, 4.5, 2.3-3.8. Analysis calculated for C₂₂ H₂₉ N₈ IOS.1/2H₂ O:C, 42.51: H, 4.86., N, 18.01; S, 10.31. Found: C, 42.70; H, 4.77; N,17.74; S, 10.42.

EXAMPLE 501-{{4'-{β-[N-(1"-methyl-1",4"-dihydropyridin-3"-yl)carbonylamino]ethyl}phenyl}}propane-1,2-dionebis(4-methylthiosemicarbazone), hydrated with 1/4 mole H₂ O (Compound223 of Scheme 32)

The quaternary salt 222 (104 mg, 0.17 mmol) in ice-cold deaerated water(30 mL) is treated with sodium bicarbonate (140 mg, 1.7 mmol) and sodiumdithionite (30 mg, 1.7 mmol). Ethyl acetate (50 mL) is added to thestirred solution, and nitrogen gas (scrubbed free of oxygen by passingthrough a basic pyrogallol solution) is bubbled through the reationmixture. After 45 minutes, the organic and aqueous layers are separated,and the aqueous layer is re-extractd with ethyl acetate (30 mL). Thecombined organic layers are dried over magnesium sulfate and the volumeof solvent is reduced to half by evaporation in vacuo. The product iseluted through a short column of neutral alumina (Aldrich, 150 mesh,Brockman 1). Evaporation of the solvent affords the dihydro derivative223 as a yellow powder. Yield 57 mg (70%). The product darkens at 130°C. and decomposes at 185° C. ¹ HNMR (CDCl₃ /DMSOd₆) δ8.0-8.3, 7.1- 7.5,6.95, 6.0-6.4, 5.6-5.8, 4.5-4.8, 3.4-3.7, 3.2, 2.8-3.4, 2.3. Analysiscalculated for C₂₂ H₃₀ N₈ OS₂.1/4H₂ O: C, 53.80; H, 6.41; N, 22.82; S,13.04. Found: C, 53.80; H, 6.27; N, 22.63; S, 13.02.

The foregoing reaction schemes and examples illustrate the preparationof a wide variety of derivatives of this invention in which thedihydropyridine⃡pyridinium salt redox carrier moieties can be one of the[DHC]/[QC⁺ ] groupings depicted on pages 19 to 38 hereinabove wherein pis zero. The preparation of yet other derivatives of this type will bereadily apparent to those skilled in the art from the teachingshereinabove, particularly in light of the illustrative synthetic methodsdetailed in the PCT application referred to hereinabove, i.e.PCT/US83/00725. Moreover, it is possible to adapt the methods ofPCT/US83/00725 and of the present specification to the preparation ofthe instant derivatives containing the carrier moieties depicted onpages 19 to 38 above wherein p=1 or 2.

Some illustrative methods for preparing the compounds of this inventionin which the carrier comprises a ##STR61## group as depicted hereinabovewherein p=1 or 2 are set forth below. It should be noted that just asthe p=1 or 2 derivatives can be made by methods analogous to thosedepicted in the reaction schemes for the p=0 derivatives, so, too, thep=0 derivatives can be prepared by methods analogous to thosespecifically described below for the P=1 or 2 derivaties. The methodsdescribed below must of course be adapted to the particular chelatingagent selected for derivation, in analogous fashion to the reactionschemes depicted above.

ILLUSTRATIVE SYNTHETIC METHODS I. Methods for Derivatizing --NH₂ or--NH--Functions Method A

The chelating agent or its protected counterpart (e.g. 74 in Scheme 9 or192 in Scheme 24 or 221 in Scheme 32) is reacted with nicotinuric acidchloride, with nicotinuric acid anhydride, or with nicotinuric acid inthe presence of a suitable coupling agent such asdicyclohexylcarbodiimide, in an appropriate organic solvent, to affordthe corresponding glycylnicotinamide, or nicotinuramide. Thenicotinuramide is then quaternized, typically by treatment with methyliodide in a suitable organic solvent, to afford the quaternaryderivative, which is then de-protected if necessary and reduced bytreatment with sodium dithionite or sodium borohydride as generallydescribed hereinabove.

Alternatively, glycine may be first reacted with a reagent capable ofintroducing an amino protecting group such as benzyloxycarbonyl ort-butoxycarbonyl and the N-protected glycine then reacted with thechelating agent or its protected counterpart in the presence of acoupling agent such as dicyclohexylcarbodiimide, followed by removal ofthe N-protecting group, followed by reaction with nicotinoyl chloride ornicotinic anhydride, or with nicotinic acid in the presence ofdicyclohexylcarbodiimide or other suitable coupling agent, to afford thenicotinuramide. The nicotinuramide may then be quaternized and thequaternary de-protected if necessary and reduced as described in thepreceding paragraph.

The procedure of the second paragraph of this method may be repeatedusing picolinic acid or its acid chloride or anhydride, or isonicotinicacid or its acid chloride or anhydride, in place of nicotinic acid orits acid chloride or anhydride, respectively, to convert chelatingagents or their protected counterparts to the corresponding glycylpicolinamides and glycyl isonicotinamides and then to the correspondingquaternary and dihydro derivatives. The procedure of the first paragraphof this method may be similarly adapted. Moreover, any of theseprocedures may be repeated, substituting a different amino acid ornicotinic acid derivative thereof for the glycine or nicotinuric acidused above, e.g. replacing glycine with alanine, valine, leucine,phenylalanine, isoleucine, methionine, asparagine or glutamine.

Alternatively, the chelating agent or its protected counterpart may bereacted with an activated ester of nicotinuric acid or the like, e.g. asucinimidyl ester such as ##STR62## and the product quaternized,de-protected if necessary and then reduced as described in the firstparagraph of this method to afford the identical products. As yetanother and highly desirable alternative, the activated ester, e.g. thesiccinimidyl ester depicted above, may be quaternized (e.g. by treatmentwith methyl iodide) and the quaternized activated ester then reactedwith the drug. The quaternary compound thus obtained may then bede-protected if necessary and reduced as described in the firstparagraph of this method.

Method B

This method is of particular use when the -NH-function is part of anamide or imide or a very low pKa primary or secondary amine.

The chelating agent (e.g. 52 in Scheme 7) is first reacted with analdehyde [e.g. formaldehyde, benzaldehyde, acetaldehyde or chloral (Cl₃CCHO)]; for example, in the case of formaldehyde, one converts the-NH-function to a ##STR63## function and thus forms a suitable bridginggroup. The resultant compound is then reacted with nicotinuric acid inthe presence of a suitable dehydrating agent, or with nicotinuric acidchloride or nicotinuric acid anhydride, to form the correspondingnicotinuric acid ester of the partial formula ##STR64## The resultantintermediate is then quaternized and reduced as in Method A. Thealternative process utilizing an activated ester or quaternaryderivative thereof which is described in Method A may be utilized toadvantage here as well.

Alternatively, the steps subsequent to formation of the ##STR65##function may be replaced with steps analogous to those detailed in thesecond paragraph of Method A.

The procedure of the preceding paragraph may be repeated using picolinicacid or its acid chloride or anhydride, or isonicotinic acid or its acidchloride or anhydride, in place of nitotinic acid or its acid chlorideor anhydride, respectively (as called for in the second paragraph ofMethod A), to convert chelating agents to the corresponding glycylpicolinic acid esters and glycyl isonicotinic acid esters and then tothe corresponding compounds of this invention. Derivatives of aminoacids other than glycine may be similarly prepared. See Method A, lastparagraph.

As yet another alternative, the intermediate compound containing the##STR66## group or the like may be reacted with thionyl chloride toafford the corresponding compound containing a ##STR67## or similargroup. That derivative may then be reacted with a metallic salt(especially a silver or thallous salt) of nicotinuric acid or the like(formed, e.g. by reacting nicotinuric acid or the like with fresh silverhydroxide or oxide or with thallous ethoxide). The resultant nicotinuricacid ester of the partial formula ##STR68## or like derivative is thenquaternized and subsequently reduced as in Method A.

Method C

The procedure of the second paragraph of Method A is followed, exceptthat removal of the N-protecting group is followed by reaction with3-quinolinecarboxylic acid or its acid chloride or anhydride instead ofnicotinic acid or its acid chloride or anhydride.

The procedure of the first paragraph of Method A may be similarlyadapted to the procedure of the 3-quinolinecarboxylic acid derivatves.Moreover, Method C may be combined with Method to afford thecorresponding 3-quinolinecarboxylic acid derivatives of the type ofchelating agent used in that method.

The procedure of the first paragraph of this method may be repeatedusing 4-isoquinolinecarboxylic acid or its acid chloride or anhydride toconvert chelating agents such as those mentioned with Methods A and B tothe corresponding 4-isoquinolinecarboxylic acid derivatives.

The procedure of the first or third paragraph of this method may berepeated, substituting a different amino acid, e.g. alanine, valine,leucine, phenylalanine, isoleucine, methionine, asparagine orglutamine-ne, for the glycine used in the first step. (See Method A,second paragraph).

The general procedures described above may be utilized to provide the1,2-dihydro derivatives as well as the 1,4-dihydros.

Method D

The procedure of the second paragraph of Method A is followed, exceptthat a reactant of the formula ##STR69## is used place of nicotinicacid. (That starting material may be prepared by reacting nicotinicanhydride, nicotinoyl chloride or nicotinic acid with glycolic acid.)

The foregoing procedure can be repeated using picolinic acid or its acidchloride or anhydride, or isonicotinic acid or its acid chloride oranhydride, in place of nicotinic acid or its acid chloride or anhydride,respectively, in the preparation of the reactant depicted above. Thisvariation affords a reactant of the formula ##STR70## which can then beused in place of nicotinic acid to prepare derivatives of chelatingagents or their protected counterparts such as those mentioned withMethod A.

Method E

The procedure of the second paragraph of Method A is followed, exceptthat a reactant of the formula ##STR71## wherein n=1-3, preferably 2, isused nicotinic acid. (That reactant may be prepared from nicotinamide,e.g. when n=2, by reacting 3-iodopropionic acid with nicotinamide.) Thequaternary salt thus obtained may then be de-protected if necessary andreduced as described in Method A. See also Scheme 26.

The procedure described above can be repeated using picolinamide orisonicotinamide in place of nicotinamide in the preparation of thereactant depicted above. This variation affords a reactant of theformula ##STR72## which can then be used in place of nicotinic acid inthe procedure of the first paragraph of this method.

II. Methods for Derivatizing --OH Functions Method F

The chelating agent or its protected counterpart (e.g. 81 of Scheme 10,or the corresponding bisthiazolidine) is reacted with nicotinuric acidchloride, with nicotinuric acid anhydride, or with nicotinuric acid inthe presence of a suitable coupling agent such asdicyclohexylcarbodiimide, in an appropriate organic solvent, to affordthe corresponding glycylnicotinate, or nicotinurate. The nicotinurate isthen quaternized, de-protected if necessary and subsequently reduced asdescribed above in Method A. The alternative process utilizing anactivated ester or quaternary derivative thereof which is described inMethod A may be utilized to advantage here as well.

Alternatively, glycine may be first reacted with a reagent capable ofintroducing an amino protecting group such as benzyloxycarbonyl ort-butylcarbonyl and the N- protected glycine then reacted with thechelating agent or its protected counterpart in the presence of acoupling agent such as dicyclohexylcarbodiimide, followed by removal ofthe N- protecting group, followed by reaction with nicotinoyl chlorideor nicotinic anhydride, or with nicotinic acid in the presence ofdicyclohexylcarbodiimide or other suitable coupling agent, to afford thenicotinurate. The nicotinurate may then be quaternized, de-protected ifnecessary and the quaternary reduced as described in the precedingparagraph.

The procedure of the second paragraph of this method may be repeatedusing picolinic acid or its acid chloride or anhydride, or isonicotinicacid or its acid chloride or anhydride, in place of nicotinic acid orits acid chloride or anhydride, respectively, to convert chelatingagents to the corresponding glycyl picolinic acid esters or glycylisonicotinic acid esters and then to the corresponding compounds of theinvention. The procedure of the first paragraph of this method may besimilarly adapted. Moreover, any of these procedures may be repeated,substituting a different amino acid or nicotinic acid derivative thereoffor the glycine or nicotinuric acid used above, e.g. replacing glycinewith alanine, valine, leucine, phenylalanine. isoleucine, methionine,asparagine or glutamine.

Method G

The procedure of the second paragraph of Method F is followed, exceptthat a reactant of the formula ##STR73## wherein n=1-3, preferably 2(prepared as described in Method E), is used in place of nicotinic acid.The quaternary salt thus obtained may then be de-protected if necessaryand reduced as described in Method A.

Method G is of particular use in preparing derivatives of chelatingagents in which the hydroxy function is hindered.

Alternatively, Method G may follow Method F, second paragraph, exceptthat it employs a reactant of the formula ##STR74## (prepared asdescribed in Method E) in place of nicotinic acid.

The procedures of this method may be repeated, substituting a differentamino acid, e.g. alanine, valine, leucine, phenylalanine, isoleucine,methionine, asparagine or glutamine, for the glycine used in the firststep. (See Method A, second paragraph).

Method H

The procedure of Method F, second paragraph, is followed, except thatremoval of the N--protecting group is followed by reaction with3-quinolinecarboxylic acid or its acid chloride or anhydride instead ofnicotinic acid or its acid chloride or anhydride.

The procedure of the first paragraph of Method F may be similarlyadapted to the production of the 3-quinolinecarboxylic acid derivatives.

The procedure of Method H may be repeated using 4-isoquinolinecarboxylicacid or its acid chloride or anhydride in place of 3-quinolinecarboxylicacid or its acid chloride or anhydride.

3-Quinolinecarboxylic acid or its acid chloride or anhydride or4-isoquinolinecarboxylic acid or its acid chloride or anhydride can alsobe substituted for nicotinic acid or its acid chloride in Method B,fourth paragraph, to afford the corresponding derivatives.

The general procedures described above may be utilized to provide the1,2-dihydro derivatives as well as the 1,4-dihydros.

Method I

The procedure of the second paragraph of Method F is followed, exceptthat a reactant of the formula ##STR75## is used in place of nicotinicacid.

A starting material of the formula set forth immediately above can alsobe substituted for nicotinic acid in Method B, paragraph 4, to affordthe corresponding derivatives.

Alternatively, Method I may follow Method F, second paragraph, exceptthat it employs a reactant of the formula ##STR76## (prepared asdescribed in Method D). These alternative Method I starting materialsmay be substituted for nicotinic acid in Method B, fourth paragraph, togive the corresponding derivatives.

The procedure of the first or third paragraph of this method may berepeated, substituting a different amino acid, e.g. alanine, valine,leucine, phenylalanine, isoleucine, methionine, asparagine or glutamine,for the glycine used in the first step. (See Method A, secondparagraph).

III. Methods for Derivatizing --COOH Functions Method J

Nicotinuric acid (N-nicotinoylglycine) or an activated ester thereof isreacted with an aminoalkanol

    H.sub.2 N--Z'--OH

wherein Z' is C₁ -C₈ straight or branched alkylene, e.g. 2-aminoethanol,to afford the corresponding intermediate alcohol, e.g. in the case of2-aminoethanol, an intermediate of the formula ##STR77## That alcohol isthen reacted with a chelating agent containing one or more --COOHfunctions, in the presence of a suitable coupling agent such asdicyclohexylcarbodiimide. The compound thus obtained is then quaternizedand subsequently reduced as described above in Method A.

Nicotinuric acid is commercially available. However, it and analogousstarting materials can be readily prepared by reacting the selectedamino acid with the acid chloride of nicotinic acid, of picolinic acid,of isonicotinic acid, of 3-quinolinecarboxylic acid, of4-isoquinolinecarboxylic acid or the like to afford the desiredN-substituted amino acid, which can then be reacted with an aminoalkanolas described above.

Method K

The chelating agent is first reacted with ethylene glycol (or otherdihydroxyalkanol having up to 8 carbon atoms), in the presence of asuitable coupling agent such as dicyclohexylcarbodiimide, to convert the--COOH function(s) to the corresponding ##STR78## Then, a N-protectedamino acid, such as N-benzyloxycarbonylglycine, which has been preparedas described in Method A, is reacted therewith in the presence ofdicyclohexylcarbodiimide or other appropriate coupling agent. Removal ofthe protecting group, e.g. by catalytic hydrogenation, affords aderivative of the chelating agent in which the original --COOH group(s)has/have, in the case of utilizing ethylene glycol and glycine, beenconverted to the structure ##STR79## That intermediate is then reactedwith a compound of the formula ##STR80## or the like, prepared asdescribed in Method E, in the presence of a coupling agent such asdicyclohexylcarbodiimide, to give the desired quaternary derivative.Subsequent reduction to the corresponding dihydro derivative proceeds asdescribed in Method A.

The procedure described above may be repeated utilizing a reactant ofthe formula ##STR81## or the like, prepared as described in Method E, inplace of the intermediate of the formula ##STR82##

Method L

A chelating agent containing one --COOH function is reacted with anequivalent amount of inositol, in the presence ofdicyclohexylcarbodiimide or other suitable coupling agent, to convertthe --COOH function to a group of the structure ##STR83## Reaction ofthat intermediate with nicotinuric acid, in the presence of a suitablecoupling agent, or with an activated ester of nicotinuric acid, affordsan intermediate in which the original --COOH has been converted to##STR84## wherein each R is H or ##STR85## the number of originalhydroxy groups esterified varying with the amount of nicotinuric acidemployed. Subsequent quaternization and reduction are carried out as inMethod A.

Alternatively, the above procedure may be repeated, replacingnicotinuric acid with an analogous starting material, prepared byreacting the selected amino acid with the acid chloride of nicotinicacid, of picolinic acid, or isonicotinic acid, of 3-quinolinecarboxylicacid, of 4-isoquinolinecarboxylic acid or the like. Repetition of theprocedure of the first paragraph of this method utilizing a greateramount of the chelating agent (e.g. 2 to 5 or more moles per mole ofinositol) provides an intermediate containing from 2 to acid residuesand from 4 to 1 hydroxyl groups. That intermediate is then reacted withnicotinuric acid to convert at least one hydroxyl group to thecorresponding ##STR86## group. Subsequent formation of the quaternaryand reduction proceed as in Method A.

Method M

The chelating agent is first reacted with 1,2-propylene glycol (or otherdihydroxyalkanol having up to 8 carbon atoms), in the presence of asuitable coupling agent such as dicyclohexylcarbodiimide, to convert the--COOH function(s) to the corresponding ##STR87## The resultantintermediate is then reacted with nicotinuric acid, in the presence ofan appropriate coupling agent, or with an activated ester of nicotinuricacid, to give an intermediate of the partial formula ##STR88##Subsequent quaternization and reduction are carried out as in Method A.

Alternatively, the above procedure may be repeated, replacingnicotinuric acid with an analogous starting material, prepared byreacting the selected amino acid with the acid chloride of nicotinicacid, of picolinic acid, of isonicotinic acid, of 3-quinolinecarboxylicacid, of 4-isoquinolinecarboxlic acid or the like.

Method N

Glucosamine, of the structural formula ##STR89## is reacted withnicotinuric acid, using equimolar amounts of the reactants, in thepresence of a suitable coupling agent such as dicyclohexylcarbodiimide,or with an activated ester of nicotinuric acid. The resultantintermediate of the formula ##STR90## is then reacted with a chelatingagent containing one reactive --COOH function, in the presence ofdicyclohexylcarbodiimide or other appropriate coupling agent, replacingone or more of the hydroxy groups with acid residue(s), the number ofgroups replaced varying with the relative amounts of reactants used.

Alternatively, the above procedure may be repeated, replacingnicotinuric acid with an analogous starting material, prepared byreacting the selected amino acid with the acid chloride of nicotinicacid, of picolinic acid, of isonicotinic acid, of 3-quinolinecarboxylicacid, of 4-isoquinolinecarboxylic acid or the like.

Suitable nontoxic pharmaceutically acceptable diluents or vehicles foruse with the present complexes of formula (III) will be apparent tothose skilled in this art. See for example. Remington's PharmaceuticalSciences, 4th Edition (1970). Obviously, the choice of suitable diluentsor vehicles will depend upon the exact nature of the particular dosageform selected.

The dosage ranges for administration of the complexes according to thisinvention will vary with the size and species of the subject, theobjective for which the complex is administered, the particular dosageform employed, and the like, as discussed below. The quantity of givendosage form needed to deliver the desired dose of theradiopharmaceutical, of course, depends upon the concentration of thecomplex in any given pharmaceutical composition/dosage form thereof andthe radioactivity thereof.

By way of example only, 5-50 mg/kg dose of formula (III)radiopharmaceutical, injected into the tail vein or carotid vein ofrats, due to the "lock in" mechanism will exhibit a very significantdifference between brain and peripheral levels of radioactivity withconsequent ready radioimaging of the brain; imaging at approximately 60to 90 minutes after administration will be most effective, since it willtake advantage of this brain/peripheral differential.

The instant radiopharmaceuticals are generally administeredintravenously. Sustained release administration, typically by slowintravenous infusion, will further enhance the site-specificity of theinstant redox system. The rate of release of the formula (III)radiopharmaceutical from the sustained release system should becomparable to the rate of in vivo oxidation of the dihydro form (III) tothe quaternary form (IV) in order to achieve the greatest degree ofenhancement of specificity.

In a further aspect, the present invention also provides a process forthe manufacture of a diagnostic agent for the visualization of an organsuch as the brain. To that end, the blood-brain barrier penetratingform, formula (III), is admixed with an aqueous buffer medium having apH value of about 4 to about 8 preferably of about 6.5 to about 7.5 inan effective radioimaging amount.

Preparation of the radiopharmaceutical can be carried out in thehospital or like location where the patient is found in order tominimize losses of radioactivity caused by the decay of the radioactivemetal. Inasmuch as the preparation for visualization is injectable, itmust be sterile and pyrogen-free; preferably, it is also isotonic. Tothis end, a so-called labeling kit can be provided that permits asimple, rapid and safe labeling of the solution to be injected with theradioactive metal. e.g., technetium-99m. Such kits are especiallydesirable when a short-lived radioisotope such as technetium 99-m isused.

The kit includes a collecting vial for receiving and/or containing anaqueous medium in which the complexing reaction can be effected.Additionally, the kit includes the chelating agent of formula (II) orchelating agent precursor of formula (I) and a pharmacologicallyacceptable reducing agent for reducing the radioactive element to anappropriate oxidation state for complexing with the chelating agent [andalso for reducing the pyridinium carrier moiety to the correspondingdihydropyridine form, when a chelating agent precursor of formula (I) ispresent].

In the case of technetium-99m, the radioactive element is received froma radionuclide generator as an aqueous pertechnetate (TcO₄) solutionsuch as an eluate in isotonic saline, as is well-known in the art. Theamount of Tc-99m required to produce a quantity of formula (III)radiopharmaceutical sufficient for diagnostic purposes is generally from0.01 milliCurie (mCi) to about 500 mCi per ml of 99mpertechnetatesolution. The reducing agent for the pertechnetate can be a thiosulfateor dithionite if the reducing reaction is to be carried out in a basicmedium, or a tin (II) salt such as SnCl₂ if the reducing reaction is tobe carried out in an acid medium.

A kit for preparing an injectable radiopharmaceutical. e.g., forcomplexing an organ-specific agent labeled with a radioactive metal,includes, in separate containers: (1) a biologically compatible, sterileaqueous medium suitable for complex formation with a radioactive metal,(2) a dihydropyridine⃡pyridinium salt carrier-containing complexing agentof formula (I) or (II) compatible therewith, and (3) a pharmaceuticaIlyacceptable reducing agent for the radioactive metal.

The dihydropyridine⃡pyridinium salt carrier moiety may be present in thekit either in its oxidized or its reduced state, as desired. Thereducing agent for the radioactive metal can be selected to reduce alsothe oxidized carrier moiety, if present, as the radioactive metal isreduced to form the complex preparatory to injection of theradiopharmaceutical into a test animal or a patient. In a preferredembodiment of this invention, a reducing agent capable of reducing boththe oxidized form of the carrier moiety and the radioactive metal ischosen and the chelating agent precursor of formula (I) is present inthe kit. In an especially preferred embodiment the kit comprises, inseparate containers (preferably aseptically and hermetically sealedvials of approximately 5-25 ml volume). (1 ) a biologically compatiblesterile aqueous medium, (2) a chelating agent precursor of formula (I),(3) a pharmacologically acceptable reducing agent capable of reducingthe chelating agent precursor of formula (I) to a chelating agent offormula (II) and also capable of reducing the radioactive metal to anoxidation state in which it is capable of complexing with the formula(II) chelating agent to form a radiopharmaceutical of formula (III).Most preferably, the reducing agent is sodium dithionite; also mostpreferably, the radioactive metal is technetium. The dithionitereduction is preferably carried out in basic medium; this may beaccomplished by providing that the aqueous medium (1) above is of basicpH,

or by adding an appropriate base (e.g. NaOH, Na₂ CO₃) when combining thekit components and the pertechnetate solution. As yet anotheralternative, the kit could comprise only two separate components: (1)the biologically compatible, sterile aqueous medium of essentiallyneutral pH containing the chelating agent precursor of formula (I); and(2) the reducing agent and the base, e.g. sodium dithionite and sodiumcarbonate.

Radioactive metal ions are typically not provided with the kit due tothe relatively short half-lives of commonly utilized radionuclides.Rather, the radionuclide is provided separately as described earlier.and admixed with the components of the kit shortly before use, as isknown for other radiopharmaceutical delivery systems. In the case oftechnetium-99m , the pertechnetate solution and the basic aqueous mediummay be first combined and then heated, e.g. from 40 to 95° C. for 10 to20 minutes, in the presence of the reducing agent then cooled to aboutroom temperature or below prior to addition of the formula (I)precursor. In this instance, the technetium will be reduced prior toreduction of the quaternary moiety to the corresponding dihydro form inwhich case a substantial portion of the quaternary salt (I) will likelychelate with the reduced technetium to form the quaternary complex (IV)in the reaction mixture as an intermediate to the dihydro complex (III).rather than the quaternary salt (I) being first converted to the dihydrochelating agent (II) and then to the dihydro complex (III).Alternatively, if only minimal or no heating is done, the precursor maybe present in the initial mixture made from the kit, and it is likely inthis instance that the formula (I) quaternary will be first reduced tothe formula (II) dihydro, which will then chelate with the reducedtechnetium to form the complex (III). If the mixture is mildly basic,e.g. pH 8 to 9, it may be administered as is, after the reduction andchelation have occurred to form the formula (III) radiopharmaceutical,or the pH may be adjusted to about 7 If the mixture is more stronglybasic, e.g. pH 13, it is generally desirable to adjust the pH to aslightly alkaline or neutral value.

Whatever the exact configuration of the kit, it is preferable for it tocontain excess chelating agent precursor (I) or chelating agent (II)with respect to the radionuclide to be complexed therewith, e.g. a 1:2molar excess The reducing agent is present in a large excess withrespect to the chelating agent precursor (I), e.g. 1:5 to 1:10. When thechelating agent (II) rather than the precursor (I) is present, then thereducing agent is preferably present in a slight excess with respect tothe radionuclide.

To effect visualization, the diagnostic agent is administered to apatient, typically intravenously, with or without further dilution by acarrier vehicle such as physiological saline. phosphate-buffered saline.plasma, or the like. Generally, the unit dose to be administered has aradioactivity of about 0.01 milliCurie (mCi) to about 100 milliCuries,preferably about 1 mCi to about 20 mCi. The solution to be injected intoan adult patient per unit dosage is about 0.01 milliliter (ml) to about1 milliliter.

After intravenous administration, imaging of the organ in vivo can takeplace after a few minutes. If desired, imaging can also take place hoursafter the injection, depending upon the half-life of the radioactivematerial that has been introduced into the patient and upon the amountof such material introduced preferably, imaging takes place 60 to 90minutes after intravenous administration.

Any conventional method of imaging for diagnostic purposes can beutilized when practicing the present invention.

In summary, then, in its broadest aspects the present invention can beseen to provide compositions of matter comprising: (1) the residue of achelating agent having at least one reactive functional group selectedfrom the group consisting of amino, carboxyl, hydroxyl, amide and imide,said functional group being not essential for the complexing propertiesof said chelating agent, said residue being characterized by the absenceof a hydrogen atom from at least one of said reactive functional groupsof said chelating agent said chelating agent being either (a) capable ofchelating with a metallic radionuclide or (b) chelated with a metallicradionuclide; and (2) a dihydropyridine⃡pyridinium salt redox carriermoiety; said chelating agent residue and said carrier moiety beingcoupled to each other to form a hydrolytically cleavable linkagebetween.

While the invention has been described in terms of various preferredembodiments, the skilled artisan will appreciate that variousmodifications, substitutions, omissions, and changes may be made withoutdeparting from the spirit thereof. Accordingly, it is intended that thescope of the present invention be limited solely by the scope of thefollowing claims.

What is claimed is:
 1. A salt having the structural formula ##STR91##wherein X⁻ is the anion of a pharmaceutically acceptable organic orinorganic acid; n is the valence of the acid anion; m, is a number whichwhen multiplied by n is equal to one; and [QC⁺ ] is a radical of theformula ##STR92## wherein the alkylene group can be straight or branchedand can contain 1 to 3 carbon atoms; R_(o) is hydrogen, methyl,##STR93## p is 0, 1 or 2, provided that, when p is 2, then the alkylenegroups can be the same or different and the R_(o) radicals can be thesame or different; R₁ is C₁ -C₇ alkyl, C₁ -C₇ haloalkyl or C₇ -C₁₀aralkyl; R₃ is C₁ to C₃ alkylene; X is --CONR'R" wherein R' and R",which can be the same or different, are each H or C₁ -C₇ alkyl, or X is--CH═NOR'" wherein R'" is H or C₁ -C₇ alkyl; and the carbonyl-containinggroupings in formulas (a) and (c) and the X substituent in formula (b)can each be attached at the 2, 3 or 4 position of the pyridinium ring.2. A salt as defined by claim 1, wherein the carbonyl-containinggrouping in formula (a) is located in the 3 position of the pyridiniumring, the X substituent in formula (b) is located in the 3 position ofthe pyridinium ring and the carbonyl-containing grouping in formula (c)is located in the 3 position of the pyridinium ring.
 3. A salt asdefined in claim 1, wherein p is zero.
 4. A salt as defined in claim 1,wherein R₁ is methyl.
 5. A salt as defined in claim 1, wherein [QC⁺ ] isa radical of formula (a).
 6. A salt as defined in claim 5, wherein [QC⁺] is a radical of formula ##STR94##
 7. A salt having the structuralformula ##STR95##